Chimeric fibroblast growth factor 19 proteins and methods of use

ABSTRACT

The present invention relates to a chimeric protein that includes an N-terminus coupled to a C-terminus, where the N-terminus includes a portion of a paracrine fibroblast growth factor (“FGF”) and the C-terminus includes a C-terminal portion of an FGF19 molecule. The portion of the paracrine FGF is modified to decrease binding affinity for heparin and/or heparan sulfate compared to the portion without the modification. The present invention also relates to pharmaceutical compositions including chimeric proteins according to the present invention, methods for treating a subject suffering from diabetes, obesity, or metabolic syndrome, and methods of screening for compounds with enhanced binding affinity for the βKlotho-FGF receptor complex involving the use of chimeric proteins of the present invention.

This application is a divisional application of U.S. patent applicationSer. No. 13/838,350, filed Mar. 15, 2013, which claims priority benefitof U.S. Provisional Patent Application No. 61/656,871, filed Jun. 7,2012, and U.S. Provisional Patent Application No. 61/664,085, filed Jun.25, 2012, each of which is hereby incorporated by reference in itsentirety.

This invention was made with government support under grant numbersDE13686, DK077276, AG019712, DK091392, and DK067158 awarded by the U.S.National Institutes of Health. The government has certain rights in thisinvention.

FIELD OF THE INVENTION

The present invention relates to chimeric fibroblast growth factor(“FGF”) proteins and uses thereof.

BACKGROUND OF THE INVENTION

Type 2 diabetes is a chronic progressive disorder, which results fromend-organ resistance to the action of insulin in combination withinsufficient insulin secretion from the pancreas. The metabolicabnormalities associated with insulin resistance and secretory defects,in particular the hyperglycemia, lead over the course of years toextensive irreversible damage to multiple organs including heart, bloodvessels, kidney, and eye. Currently, nearly 200 million or 2.9% of theworld population have type 2 diabetes (World Health Organization,Diabetes Fact Sheet No 312, January 2011; Wild et al., “GlobalPrevalence of Diabetes: Estimates for the Year 2000 and Projections for2030,” Diabetes Care 27(5):1047-1053 (2004)), and its prevalence isrising at an alarmingly fast pace in parallel with the rise in theprevalence of overweight and obesity (World Health Organization, Obesityand Overweight Fact Sheet No 311, January 2011). Until the end of the20^(th) century, type 2 diabetes was observed only in adults but whatwas once known as “adult-onset diabetes” is now also diagnosed inchildren and adolescents, and this growing incidence can be related tothe increase in overweight and obesity among children and adolescents.The prevalence of pre-diabetes, an intermediate metabolic stage betweennormal glucose homeostasis and diabetes, is even greater than that oftype 2 diabetes. Currently, nearly 80 million or 26% of the populationin the United States alone have pre-diabetes (Center for Disease Controland Prevention, National Diabetes Fact Sheet 2011), and as such are athigh risk for progressing to type 2 diabetes. Type 2 diabetes ranksamong the ten leading causes of death worldwide, and the World HealthOrganization projects that mortality from diabetes (90% of which is type2) will more than double within the next decade (World HealthOrganization, Diabetes Fact Sheet No 312, January 2011). Type 2 diabetesalso is a major cause of disability. As a consequence of diabeticretinopathy, about 10% of all patients with diabetes in the worlddevelop severe visual impairment and 2% become blind 15 years into thedisease (World Health Organization, Diabetes Fact Sheet No 312, January2011). Diabetic neuropathy, which affects up to half of all patientswith diabetes worldwide (World Health Organization, Diabetes Fact SheetNo 312, January 2011), accounts for the majority of nontraumaticlower-limb amputations. Indeed, in its recently published firstworldwide report on non-infectious diseases, the World HealthOrganization considers diabetes, together with other chronicnon-infectious diseases like cancer and heart disease, a global economicand social burden, which exceeds that imposed by infectious diseasessuch as HIV/AIDS.

The current drug therapy for type 2 diabetes is focused on correctingthe hyperglycemia in the patients. Although a number of small moleculesand biologics with different mechanisms of anti-hyperglycemic action areavailable for use as mono-therapy or combination therapy, most, if notall of these have limited efficacy, limited tolerability, andsignificant adverse effects (Moller, “New Drug Targets for Type 2Diabetes and the Metabolic Syndrome,” Nature 414(6865):821-827 (2001)).For example, treatment with sulfonylureas, glinides, thiazolidinediones,or insulin has been associated with weight gain, which is an undesiredeffect since overweight is considered a driving force in thepathogenesis of type 2 diabetes. Some of these treatments have also beenassociated with increased risk of hypoglycemia. A limitation specific tothe thiazolidinediones is the potential for adverse cardiovasculareffects (DeSouza et al., “Therapeutic Targets to Reduce CardiovascularDisease in Type 2 Diabetes,” Nat Rev Drug Discov 8(5):361-367 (2009)). Ameta-analysis of clinical data on the thiazolidinedione rosiglitazone(Avandia®), which was widely used for the treatment of type 2 diabetes,found that the drug increased the risk of myocardial infarction inpatients with type 2 diabetes (Nissen et al., “Effect of Rosiglitazoneon the Risk of Myocardial Infarction and Death from CardiovascularCauses,” N Engl J Med 356(24):2457-2471 (2007)). Of all diabeticcomplications, cardiovascular disease is the main cause of morbidity andmortality in patients with diabetes (World Health Organization, DiabetesFact Sheet No 312, January 2011; Center for Disease Control andPrevention, National Diabetes Fact Sheet 2011), and hence an aggravationof cardiovascular risk by drug treatment is absolutely unacceptable. Inthe wake of the debate about the cardiovascular safety ofthiazolidinediones, the FDA issued a guidance on evaluatingcardiovascular risk in new anti-diabetic therapies to treat type 2diabetes (Opar A, “Diabetes Drugs Pass Cardiovascular Risk Check,” NatRev Drug Discov 8(5):343-344 (2009)). Meanwhile, thiazolidinediones losttheir popularity. Even for glucagon-like peptide-1 agonists, one of thelatest class of drugs introduced for the treatment of type 2 diabetes,concerns about safety have been raised, namely the potential forcarcinogenicity (Opar A, “Diabetes Drugs Pass Cardiovascular RiskCheck,” Nat Rev Drug Discov 8(5):343-344 (2009)). Therefore, noveltherapies that are more effective and safer than existing drugs areneeded. Since the currently available drugs do not directly targetcomplications of advanced diabetic disease, especially cardiovasculardisease, therapies that are not only effective in lowering blood glucosebut also reduce cardiovascular risk factors such as dyslipidemia areparticularly desired.

A search conducted by Eli Lilly & Co. for potential novelbiotherapeutics to treat type 2 diabetes led to the discovery offibroblast growth factor (FGF) 21 as a protein that stimulates glucoseuptake into adipocytes in an insulin-independent fashion (Kharitonenkovet al., “FGF-21 as a Novel Metabolic Regulator,” J Clin Invest 115(6):1627-1635 (2005)). FGF21 has since emerged as a key endocrine regulatornot only of glucose metabolism but also of lipid metabolism, and hasbecome one of the most promising drug candidates for the treatment oftype 2 diabetes, obesity, and metabolic syndrome. In mouse models ofdiabetes and obesity, pharmacologic doses of FGF21 lower plasma glucoseand increase insulin sensitivity (Kharitonenkov et al., “FGF-21 as aNovel Metabolic Regulator,” J Clin Invest 115(6):1627-1635 (2005);Coskun et al., “Fibroblast growth factor 21 corrects obesity in mice,”Endocrinology 149(12):6018-6027 (2008)). Concurrently, FGF21 lowersplasma triglyceride and cholesterol, enhances lipolysis and suppresseslipogenesis, and accelerates energy expenditure (Kharitonenkov et al.,“FGF-21 as a Novel Metabolic Regulator,” J Clin Invest 115(6):1627-1635(2005); Coskun et al., “Fibroblast growth factor 21 corrects obesity inmice,” Endocrinology 149(12):6018-6027 (2008)). In obese mice, FGF21causes weight loss, in lean mice, it is weight neutral (Kharitonenkov etal., “FGF-21 as a Novel Metabolic Regulator,” J Clin Invest115(6):1627-1635 (2005); Coskun et al., “Fibroblast growth factor 21corrects obesity in mice,” Endocrinology 149(12):6018-6027 (2008)).Thus, FGF21 has some of the most desired characteristics of a drug forthe treatment of type 2 diabetes; not only does it improve glycemiccontrol, but also directly affects cardiovascular risk factors, such ashypertriglyceridemia, and reduces obesity, which is considered thesingle most important promoter of type 2 diabetes. Importantly, FGF21does not induce hypoglycemia (Kharitonenkov et al., “FGF-21 as a NovelMetabolic Regulator,” J Clin Invest 115(6):1627-1635 (2005)), a sideeffect that can occur with several of the current anti-diabetictherapies, including insulin. Moreover, FGF21 does not exhibit anymitogenic activity in mice (Kharitonenkov et al., “FGF-21 as a NovelMetabolic Regulator,” J Clin Invest 115(6):1627-1635 (2005)), ruling outthe possibility of a carcinogenic risk. The findings on FGF21 therapy inmouse models of diabetes have been reproduced in diabetic rhesus monkeys(Kharitonenkov et al., “The Metabolic State of Diabetic Monkeys isRegulated by Fibroblast Growth Factor-21,” Endocrinology 148(2):774-781(2007)), and are currently followed up with clinical trials in humans(Kharitonenkov et al., “FGF21 Reloaded: Challenges of a Rapidly GrowingField,” Trends Endocrinol Metab 22(3):81-86 (2011)). However, there is aneed for more effective FGF21 therapeutics.

The present invention overcomes these and other deficiencies in the art.

SUMMARY OF THE INVENTION

One aspect of the present invention relates to a chimeric protein. Thechimeric protein includes an N-terminus coupled to a C-terminus, wherethe N-terminus includes a portion of a paracrine fibroblast growthfactor (“FGF”) and the C-terminus includes a C-terminal portion of anFGF19 molecule. The portion of the paracrine FGF is modified to decreasebinding affinity for heparin and/or heparan sulfate compared to theportion without the modification.

Another aspect of the present invention relates to a method for treatinga subject suffering from a disorder. This method involves selecting asubject suffering from the disorder. The method also involves providinga chimeric FGF protein, where the chimeric FGF protein includes anN-terminus coupled to a C-terminus. The N-terminus includes a portion ofa paracrine FGF and the C-terminus includes a C-terminal portion ofFGF19. The portion of the paracrine FGF is modified to decrease bindingaffinity for heparin and/or heparan sulfate compared to the portionwithout the modification. This method also involves administering atherapeutically effective amount of the chimeric FGF protein to theselected subject under conditions effective to treat the disorder.

Another aspect of the present invention relates to a method of making achimeric FGF protein possessing enhanced endocrine activity. This methodinvolves introducing one or more modifications to a FGF protein, wherethe modification decreases the affinity of the FGF protein for heparinand/or heparan sulfate and coupling a C-terminal portion of FGF19 thatincludes a βKlotho co-receptor binding domain to the modified FGFprotein's C-terminus, whereby a chimeric FGF protein possessing enhancedendocrine activity is made.

Yet another aspect of the present invention relates to a method offacilitating fibroblast growth factor receptor (“FGFR”)-βKlothoco-receptor complex formation. This method involves providing a cellthat includes a βKlotho co-receptor and an FGFR and providing a chimericFGF protein. The chimeric FGF protein includes a C-terminal portion ofFGF19 and a portion of a paracrine FGF, where the portion of theparacrine FGF is modified to decrease binding affinity for heparinand/or heparan sulfate compared to the portion without the modification.This method also involves contacting the cell and the chimeric FGFprotein under conditions effective to cause FGFR-βKlotho co-receptorcomplex formation.

Yet a further aspect of the present invention relates to a method ofscreening for agents capable of facilitating FGFR-βKlotho complexformation in the treatment of a disorder. This method involves providinga chimeric FGF that includes an N-terminus coupled to a C-terminus,where the N-terminus includes a portion of a paracrine FGF and theC-terminus includes a C-terminal portion of FGF19. The portion of theparacrine FGF is modified to decrease binding affinity for heparinand/or heparan sulfate compared to the portion without the modification.This method also involves providing a binary βKlotho-FGFR complex andproviding one or more candidate agents. This method further involvescombining the chimeric FGF, the binary βKlotho-FGFR complex, and the oneor more candidate agents under conditions permitting the formation of aternary complex between the chimeric FGF and the binary βKlotho-FGFRcomplex in the absence of the one or more candidate agents. This methodalso involves identifying the one or more candidate agents that decreaseternary complex formation between the chimeric FGF and the binaryβKlotho-FGFR complex compared to the ternary complex formation in theabsence of the one or more candidate agents as suitable for treating thedisorder.

Fibroblast growth factors (FGFs) 19, 21, and 23 are hormones thatregulate in a Klotho co-receptor-dependent fashion major metabolicprocesses such as glucose and lipid metabolism (FGF21) and phosphate andvitamin D homeostasis (FGF23). The role of heparan sulfateglycosaminoglycan in the formation of the cell surface signaling complexof endocrine FGFs has remained unclear. To decipher the role of HS inendocrine FGF signaling, we generated FGF19 and FGF23 mutant ligandsdevoid of HS binding and compared their signaling capacity with that ofwild-type ligands. The data presented herein show that the mutatedligands retain full metabolic activity demonstrating that HS does notparticipate in the formation of the endocrine FGF signaling complex.Here it is shown that heparan sulfate is not a component of the signaltransduction unit of FGF19 and FGF23. A paracrine FGF is converted intoan endocrine ligand by diminishing heparan sulfate binding affinity ofthe paracrine FGF and substituting its C-terminal tail for that of anendocrine FGF containing the Klotho co-receptor binding site in order tohome the ligand into the target tissue. The ligand conversion provides anovel strategy for engineering endocrine FGF-like molecules for thetreatment of metabolic disorders, including global epidemics such astype 2 diabetes and obesity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D are schematic diagrams showing side-by-side comparison ofthe HS-binding site of FGF2, FGF19, and FGF23, and working model of theendocrine FGF signaling complex. FIG. 1A shows interactions of FGF2(schematic representation) with a heparin hexasaccharide (shown assticks) as observed in the crystal structure of the 2:2 FGF2-FGFR1cdimer (PDB ID: 1FQ9; (Schlessinger et al., Mol. Cell 6:743-750 (2000),which is hereby incorporated by reference in its entirety)). The heparinhexasaccharide consists of three disaccharide units of 1→4 linkedN-sulfated-6-O-sulfated D-glucosamine and 2-O-sulfated L-iduronic acid.Note that the heparin hexasaccharide interacts with both side chain andbackbone atoms of residues in the HS-binding site of FGF2. Dashed linesdenote hydrogen bonds. K128, R129, and K134, which make the majority ofhydrogen bonds with the heparin hexasaccharide, are boxed. The β-strandnomenclature follows the original FGF1 and FGF2 crystal structures (Agoet al., J. Biochem. 110:360-363 (1991); Eriksson et al., Proc. Nat'l.Acad. Sci. U.S.A. 88:3441-3445 (1991); Zhang et al., Proc. Nat'l. Acad.Sci. U.S.A. 88:3446-3450 (1991); Zhu et al., Science 251:90-93 (1991),which are hereby incorporated by reference in their entirety). Pleasenote that compared to the prototypical β-trefoil fold seen in soybeantrypsin inhibitor (PDB ID: 1TIE; (Onesti et al., J. Mol. Biol.217:153-176 (1991), which is hereby incorporated by reference in itsentirety) and interleukin 1β (PDB ID: 1I1B; (Finzel et al., J. Mol.Biol. 209:779-791 (1989), which is hereby incorporated by reference inits entirety), the β10-β11 strand pairing in FGF2 and other paracrineFGFs is less well defined. FIGS. 1B and 1C show cartoon representationof the crystal structures of FGF19 (PDB ID: 2P23; (Goetz et al., Mol.Cell Biol. 27:3417-3428 (2007), which is hereby incorporated byreference in its entirety)) (FIG. 1B) and FGF23 (PDB ID: 2P39; (Goetz etal., Mol. Cell Biol. 27:3417-3428 (2007), which is hereby incorporatedby reference in its entirety)) (FIG. 1C) shown in the same orientationas the FGF2 structure in FIG. 1A. Side chains of residues that map tothe corresponding HS-binding sites of these ligands are shown as sticks.Residues selected for mutagenesis to knock out residual HS binding inFGF19 and FGF23 are boxed. NT and CT indicate N- and C-termini of theFGFs. FIG. 1D is a schematic of two working models for the endocrineFGF-FGFR-Klotho signal transduction unit. A recent study on the ternarycomplex formation between FGF21, FGFR1c and βKlotho supports the 1:2:1model rather than the 2:2:2 model (Ming et al., J. Biol. Chem.287:19997-20006 (2012), which is hereby incorporated by reference in itsentirety). For comparison, a schematic of the paracrine FGF-FGFR-HSsignaling unit is shown, which was made based on the crystal structureof the 2:2:2 FGF2-FGFR1c-HS complex (PDB ID: 1FQ9; (Schlessinger et al.,Mol. Cell 6:743-750 (2000), which is hereby incorporated by reference inits entirety)). HS engages both paracrine FGF and receptor to enhancebinding of FGF to its primary and secondary receptors thus promotingreceptor dimerization. A question mark denotes whether or not HS is alsoa component of the endocrine FGF signaling complex.

FIG. 2 shows a sequence alignment of the endocrine FGFs, FGF1, and FGF2.The amino acid sequences of the mature human FGF19, FGF21, and FGF23ligands are aligned. Also included in the alignment are the humansequences of FGF1 and FGF2, prototypical paracrine FGFs, which were usedin the experiments described herein, in which FGF1 and FGF2 wereconverted into endocrine FGF ligands. Residue numbers corresponding tothe human sequence of FGF1 (SEQ ID NO: 1) (GenBank Accession No.AAH32697, which is hereby incorporated by reference in its entirety),FGF2 (SEQ ID NO: 121) (GenBank Accession No. EAX05222, which is herebyincorporated by reference in its entirety), FGF19 (SEQ ID NO: 233)(GenBank Accession No. NP_005108, which is hereby incorporated byreference in its entirety), FGF21 (SEQ ID NO: 332) (GenBank AccessionNo. NP_061986, which is hereby incorporated by reference in itsentirety), and FGF23 (SEQ ID NO:345) (GenBank accession no. AAG09917,which is hereby incorporated by reference in its entirety) are inparenthesis to the left of the alignment. Secondary structure elementsare labeled, and residues containing these elements for known secondarystructures are boxed. Gaps (dashes) were introduced to optimize thesequence alignment. The f3-trefoil core domain for known FGF crystalstructures is shaded gray. Blue bars on top of the alignment indicatethe location of the HS-binding regions. HS-binding residues selected formutagenesis are shaded blue.

FIGS. 3A-3G show Surface plasmon resonance (“SPR”) results relating toknockout of residual heparin binding in FGF19 and FGF23 by site-directedmutagenesis. FIG. 3A shows an overlay of SPR sensorgrams illustratingheparin binding of FGF2, FGF19, FGF21, and FGF23 (left panel) and anexploded view of the binding responses for FGF19-, FGF21-, andFGF23-heparin interactions (right panel). Heparin was immobilized on abiosensor chip, and 400 nM of FGF2, FGF19, FGF21, or FGF23 were passedover the chip. Note that FGF19, FGF21, and FGF23 exhibit measurable,residual heparin binding and that differences in heparin binding existbetween these three endocrine FGFs. FIGS. 3B-3D show overlays of SPRsensorgrams illustrating binding of FGF19 to heparin (FIG. 3B) and lackof interaction between the FGF19^(K149A) mutant and heparin (FIG. 3C)and between the FGF19^(K149A, R157A) mutant and heparin (FIG. 3D).Heparin was immobilized on a biosensor chip, and increasingconcentrations of FGF19 were passed over the chip. Thereafter,FGF19^(K149A) or FGF19^(K149A, R157A) was injected over the heparin chipat the highest concentration tested for the wild-type ligand. FIGS.3E-3G show overlays of SPR sensorgrams illustrating binding of FGF23 toheparin (FIG. 3E), poor interaction between the FGF23^(R48A, N49A)mutant and heparin (FIG. 3F), and lack of interaction between theFGF23^(R140A, R143A) mutant and heparin (FIG. 3G). Heparin wasimmobilized on a biosensor chip, and increasing concentrations of FGF23were passed over the chip. FGF23^(R48A, N49A) or FGF23^(R140A, R143A)was then injected over the heparin chip at the highest concentrationtested for the wild-type ligand.

FIGS. 4A-4D show results demonstrating that HS is dispensable for themetabolic activity of FGF19 and FGF23. FIG. 4A shows results of animmunoblot analysis of phosphorylation of FRS2α (pFRS2α) and 44/42 MAPkinase (p44/42 MAPK) in H4IIE hepatoma cells following stimulation withthe FGF19^(K149A) mutant, the FGF19^(K149A, R157A) mutant, or wild-typeFGF19. Numbers above the lanes give the amounts of protein added in ngml⁻¹. Total 44/42 MAPK protein expression was used as a loading control.FIG. 4B shows results of an immunoblot analysis of phosphorylation ofFRS2α (pFRS2α) and 44/42 MAP kinase (p44/42 MAPK) in a HEK293-αKlothocell line following stimulation with the FGF23^(R48A, N49A) mutant, theFGF23^(R140A, R143A) mutant, or wild-type FGF23. Numbers above the lanesgive the amounts of protein added in ng ml⁻¹. Total 44/42 MAPK andαKlotho protein expression were used as loading controls. FIG. 4C showsgraphical results of a quantitative analysis of CYP7A1 and CYP8B1 mRNAexpression in liver tissue from mice treated with FGF19^(K149A),FGF19^(K149A, R157A), FGF19, or vehicle. 1 mg of protein per kg of bodyweight was given. Data are presented as mean±SEM; ***, P<0.001 byStudent's t test. FIG. 4D shows graphical results of analysis of serumphosphate concentrations (serum P_(i)) in mice before and 8 h afterintraperitoneal injection of FGF23^(R48A, N49A), FGF23^(R140A, R143A),FGF23, or vehicle. Wild-type mice were given a single dose of protein(0.29 mg kg body weight⁻¹), whereas Fgf23 knockout mice received twodoses of 0.71 mg kg body weight⁻¹ each. Data are presented as mean±SEM;*, P<0.05, and **, P<0.01 by ANOVA.

FIGS. 5A-5G show design and results relating to the conversion of FGF2into an endocrine ligand. FIG. 5A is a schematic of human FGF2, FGF19,FGF21, FGF23, and engineered FGF2-FGF19, FGF2-FGF21, and FGF2-FGF23chimeras. Amino acid boundaries of each ligand and of each component ofthe chimeras are labeled with residue letter and number. The β-trefoilcore domain for the known ligand crystal structures is shaded gray.HS-binding residues mutated in the FGF2 portion of chimeras are labeledwith residue letter and number. Also labeled are the arginine residuesof the proteolytic cleavage site in the C-terminal region of FGF23 thatwere mutated to glutamine in both FGF23 and the FGF2-FGF23 chimeras.FIGS. 5B and 5C show overlays of SPR sensorgrams illustrating binding ofFGF2^(WTcore)-FGF21^(C-tail) (FIG. 5B) andFGF2^(ΔHBScore)-FGF21^(C-tail) (FIG. 5C) to heparin, and fittedsaturation binding curves. Heparin was immobilized on a biosensor chip,and increasing concentrations of FGF2^(WTcore)-FGF21^(C-tail) orFGF2^(ΔHBScore)-FGF21^(C-tail) were passed over the chip. Dissociationconstants (K_(D)S) were derived from the saturation binding curves.FIGS. 5D and 5E show overlays of SPR sensorgrams illustrating binding ofFGF2^(WTcore)-FGF23^(C-tail) (FIG. 5D) andFGF2^(ΔHBScore)-FGF23^(C-tail) (FIG. 5E) to heparin. Increasingconcentrations of FGF2^(WTcore)-FGF23^(C-tail) orFGF2^(ΔHBScore)-FGF23^(C-tail) were passed over a chip containingimmobilized heparin. FIGS. 5F and 5G show results of immunoblot analysisfor Egr1 expression in HEK293 cells following stimulation with chimerasor native FGFs as denoted. Numbers above the lanes give the amounts ofprotein added in nanomolar. GAPDH protein expression was used as aloading control.

FIG. 6 is a schematic illustrating the conversion of FGF1 into anendocrine ligand. Shown are schematic drawings of human FGF1, FGF19,FGF21, FGF23, and exemplary FGF1-FGF19, FGF1-FGF21, and FGF1-FGF23chimeras according to the present invention. Amino acid boundaries ofeach ligand and of each component of the chimeras are labeled withresidue letter and number. The β-trefoil core domain for the knownligand crystal structures is shaded gray. HS-binding residues mutated inthe FGF1 portion of chimeras are labeled with residue letter and number.Also labeled are the arginine residues of the proteolytic cleavage sitein the C-terminal region of FGF23 that were mutated to glutamine in bothFGF23 and the FGF1-FGF23 chimeras.

FIGS. 7A-7G show results demonstrating that theFGF2^(ΔHBScore)-FGF23^(C-tail) chimera exhibits FGF23-like activity.FIGS. 7A and 7B show overlays of SPR sensorgrams illustrating inhibitionby FGF2^(ΔHBScore)-FGF23^(C-tail) (FIG. 7A) or FGF23 (FIG. 7B) ofαKlotho-FGFR1c binding to FGF23 immobilized on a biosensor chip.Increasing concentrations of FGF2^(ΔHBScore)-FGF23^(C-tail) or FGF23were mixed with a fixed concentration of αKlotho-FGFR1c complex, and themixtures were passed over a FGF23 chip. FIG. 7C shows an overlay of SPRsensorgrams illustrating failure of FGF2 to inhibit αKlotho-FGFR1cbinding to FGF23. FGF2 and αKlotho-FGFR1c complex were mixed at a molarratio of 15:1, and the mixture was passed over a biosensor chipcontaining immobilized FGF23. FIGS. 7D and 7E show overlays of SPRsensorgrams illustrating no inhibition by FGF2^(ΔHBScore)-FGF23^(C-tail)(FIG. 7D) or FGF23 (FIG. 7E) of βKlotho-FGFR1c binding to FGF21.FGF2^(ΔHBScore)-FGF23^(C-tail) or FGF23 were mixed with βKlotho-FGFR1ccomplex at a molar ratio of 10:1, and the mixtures were passed over abiosensor chip containing immobilized FGF21. FIG. 7F shows analysis ofserum phosphate concentrations (serum P_(i)) in mice before and 8 hafter intraperitoneal injection of FGF2^(ΔHBScore)-FGF23^(C-tail),FGF2^(WTcore)-FGF23^(C-tail), FGF23, or vehicle. Wild-type mice andαKlotho knockout mice were given 0.21 mg and 0.51 mg of protein,respectively, per kg of body weight. Data are presented as mean±SEM; **,P<0.01; ***, P<0.001 by ANOVA. FIG. 7G shows quantitative analysis ofCYP27B1 mRNA expression in renal tissue from mice injected withFGF2^(ΔHBScore)-FGF23^(C-tail), FGF2^(WTcore)-FGF23^(C-tail), FGF23, orvehicle. 0.21 mg of protein per kg of body weight were injected. Dataare presented as mean±SEM; ***, P<0.001 by ANOVA.

FIGS. 8A-8G show results demonstrating that theFGF2^(ΔHBScore)-FGF21^(C-tail) chimera exhibits FGF21-like activity.FIGS. 8A-8B show overlays of SPR sensorgrams illustrating inhibition byFGF2^(ΔHBScore)-FGF21^(C-tail) (FIG. 8A) or FGF21 (FIG. 8B) ofβKlotho-FGFR1c binding to FGF21 immobilized on a biosensor chip.Increasing concentrations of FGF2^(ΔHBScore)-FGF21^(C-tail) or FGF21were mixed with a fixed concentration of βKlotho-FGFR1c complex, and themixtures were passed over a FGF21 chip. FIG. 8C shows an overlay of SPRsensorgrams illustrating failure of FGF2 to inhibit βKlotho-FGFR1cbinding to FGF21. FGF2 and βKlotho-FGFR1c complex were mixed at a molarratio of 15:1, and the mixture was passed over a biosensor chipcontaining immobilized FGF21. FIGS. 8D-8E show overlays of SPRsensorgrams illustrating no inhibition by FGF2^(ΔHBScore)-FGF21^(C-tail)(FIG. 8D) or FGF21 (FIG. 8E) of αKlotho-FGFR1c binding to FGF23.FGF2^(ΔHBScore) FGF21^(C-tail) or FGF21 were mixed with αKlotho-FGFR1ccomplex at a molar ratio of 10:1, and the mixtures were passed over abiosensor chip containing immobilized FGF23. FIG. 8F shows results ofimmunoblot analysis for Egr1 expression in HEK293-βKlotho cellsstimulated with FGF2^(ΔHBScore)-FGF21^(C-tail) or FGF21. Numbers abovethe lanes give the amounts of protein added in ng ml⁻¹. GAPDH proteinexpression was used as a loading control. Note that theFGF2^(ΔHBScore)-FGF21^(C-tail) chimera is more potent than native FGF21at inducing Egr1 expression suggesting that the chimera has agonisticproperty. This is expected since the core domain of FGF2 has inherentlygreater binding affinity for FGFR than the core domain of FGF21 (seeFIGS. 10A and 10C). FIG. 8G shows graphical results of analysis of bloodglucose concentrations in mice before and at the indicated time pointsafter intraperitoneal injection of insulin alone, insulin plusFGF2^(ΔHBScore)-FGF21^(C-tail) chimera, insulin plus FGF21, or vehiclealone. 0.5 units of insulin per kg of body weight and 0.3 mg of FGF21ligand per kg of body weight were injected. Blood glucose concentrationsare expressed as percent of pre-injection values. Data are presented asmean±SEM.

FIGS. 9A-9C show the glucose-lowering effects in ob/ob mice of FGF1variants according to the present invention. FIG. 9A shows graphicalresults of analysis of blood glucose concentrations in ob/ob mice beforeand at the indicated time points after subcutaneous injection of FGF1 orFGF21. FIG. 9B shows graphical results of analysis of blood glucoseconcentrations in ob/ob mice before and at the indicated time pointsafter subcutaneous injection of FGF1, FGF1^(ΔNT), or FGF1^(ΔHBS). FIG.9C shows graphical results of analysis of blood glucose concentrationsin ob/ob mice before and at the indicated time points after subcutaneousinjection of FGF1 or FGF1^(ΔHBScore)-FGF21^(C-tail) chimera. For theexperiments shown in FIGS. 9A-9C, ob/ob mice were injected with a bolusof 0.5 mg of FGF protein per kg of body weight. Data are presented asmean±SD.

FIGS. 10A-10F show results demonstrating that endocrine FGFs have lowbinding affinity for FGFR1c compared to FGF2. FIGS. 10A-10D showoverlays of SPR sensorgrams illustrating binding of FGFR1c to FGF2 (FIG.10A), FGF19 (FIG. 10B), FGF21 (FIG. 10C), and FGF23 (FIG. 10D), andfitted saturation binding curves. Increasing concentrations of FGFR1cligand-binding domain were passed over a biosensor chip containingimmobilized FGF2, FGF19, FGF21, or FGF23. FIG. 10E shows an overlay ofSPR sensorgrams illustrating binding of αKlotho-FGFR1c complex to FGF23.Increasing concentrations of αKlotho-FGFR1c complex were passed over abiosensor chip containing immobilized FGF23. FIG. 8F shows an overlay ofSPR sensorgrams showing lack of interaction between the C-terminal tailpeptide of FGF23 and FGFR1c. FGF23^(C-tail) was immobilized on abiosensor chip and increasing concentrations of FGFR1c ligand-bindingdomain were passed over the chip. Dissociation constants (K_(D)s) givenin FIGS. 10A-10E were derived from the saturation binding curves.

FIG. 11 shows an alignment of the C-terminal tail sequences of humanFGF19 (SEQ ID NO: 233) (GenBank Accession No. NP_005108, which is herebyincorporated by reference in its entirety), FGF21 (SEQ ID NO: 332)(GenBank Accession No. NP_061986, which is hereby incorporated byreference in its entirety), and FGF23 (SEQ ID NO:345) (GenBank accessionno. AAG09917, which is hereby incorporated by reference in itsentirety). Residue numbers are in parenthesis to the left of thealignment. Gaps (dashes) were introduced to optimize the alignment.Residues that are identical between FGF19 and FGF21 are shaded gray.Note that 40% of these residues map to the most C-terminal sequence.

FIG. 12 shows an alignment of the C-terminal tail sequences of FGF19orthologs (including human (SEQ ID NO: 233), gorilla (SEQ ID NO: 234),chimpanzee (SEQ ID NO: 235), gibbon (SEQ ID NO: 238), rhesus monkey (SEQID NO: 236), orangutan (SEQ ID NO: 237), marmoset (SEQ ID NO: 239),mouse lemur (SEQ ID NO: 240), sloth (SEQ ID NO: 241), panda (SEQ ID NO:242), pig (SEQ ID NO: 243), bovine (SEQ ID NO: 244), dog (SEQ ID NO:245), rabbit (SEQ ID NO: 246), megabat (SEQ ID NO: 247), dolphin (SEQ IDNO: 248), microbat (SEQ ID NO: 249), platypus (SEQ ID NO: 250),opossum(SEQ ID NO: 251), anole lizard (SEQ ID NO: 252), pika (SEQ ID NO:253), guinea pig (SEQ ID NO: 254), tree shrew (SEQ ID NO: 255), rat (SEQID NO: 256), mouse (SEQ ID NO: 257), chicken (SEQ ID NO: 258), zebrafinch (SEQ ID NO: 259), zebrafish (SEQ ID NO: 260), and frog (SEQ ID NO:261)). Residue numbers are in parenthesis to the left of the alignment.Gaps (dashes) were introduced to optimize the alignment. Orthologresidues identical to human FGF19 are shaded gray.

FIG. 13 shows an alignment of the C-terminal tail sequences of humanFGF19 (SEQ ID NO:233) (GenBank Accession No. NP_005108, which is herebyincorporated by reference in its entirety), FGF21 (SEQ ID NO:332)(GenBank Accession No. NP_061986, which is hereby incorporated byreference in its entirety), and variants of FGF19 harboring a singleamino acid deletion or substitution for a residue unique to FGF21.Residue numbers for the sequences of native FGF19 and FGF21 are inparenthesis to the left of the alignment. Gaps (dashes) were introducedto optimize the alignment. In the sequence of native FGF21(SEQ IDNO:332), residues unique to FGF21 are bold and boxed, and in thesequences of the variants of the FGF19 C-terminal tail, introduced FGF21residues are also bold and boxed and deleted FGF19 residues areindicated by a dash (bold and boxed).

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the present invention relates to a chimeric protein. Thechimeric protein includes an N-terminus coupled to a C-terminus, wherethe N-terminus includes a portion of a paracrine fibroblast growthfactor (“FGF”) and the C-terminus includes a C-terminal portion of anFGF19. The portion of the paracrine FGF is modified to decrease bindingaffinity for heparin and/or heparan sulfate compared to the portionwithout the modification.

As described by Goetz et al. (Goetz et al., “Molecular Insights into theKlotho-Dependent, Endocrine Mode of Action of Fibroblast Growth Factor19 Subfamily Members,” Mol Cell Biol 3417-3428 (2007), which is herebyincorporated by reference in its entirety), the mammalian fibroblastgrowth factor (FGF) family comprises 18 polypeptides (FGF1 to FGF10 andFGF16 to FGF23), which participate in a myriad of biological processesduring embryogenesis, including but not limited to gastrulation, bodyplan formation, somitogenesis, and morphogenesis of essentially everytissue/organ such as limb, lung, brain, and kidney (Bottcher et al.,“Fibroblast Growth Factor Signaling During Early VertebrateDevelopment,” Endocr Rev 26:63-77 (2005), and Thisse et al., “Functionsand Regulations of Fibroblast Growth Factor Signaling During EmbryonicDevelopment,” Dev Biol 287:390-402 (2005), which are hereby incorporatedby reference in their entirety).

FGFs execute their biological actions by binding to, dimerizing, andactivating FGFR tyrosine kinases, which are encoded by four distinctgenes (Fgfr1 to Fgfr4). Prototypical FGFRs consist of an extracellulardomain composed of three immunoglobulin-like domains, a single-passtransmembrane domain, and an intracellular domain responsible for thetyrosine kinase activity (Mohammadi et al., “Structural Basis forFibroblast Growth Factor Receptor Activation,” Cytokine Growth FactorRev 16:107-137 (2005), which is hereby incorporated by reference in itsentirety).

The number of principal FGFRs is increased from four to seven due to amajor tissue-specific alternative splicing event in the second half ofthe immunoglobulin-like domain 3 of FGFR1 to FGFR3, which createsepithelial lineage-specific “b” and mesenchymal lineage-specific “c”isoforms (Mohammadi et al., “Structural Basis for Fibroblast GrowthFactor Receptor Activation,” Cytokine Growth Factor Rev 16:107-137(2005) and Ornitz et al., “Fibroblast Growth Factors,” Genome Biol2(3):reviews3005.1-reviews3005.12 (2001), which are hereby incorporatedby reference in their entirety). Generally, the receptor-bindingspecificity of FGFs is divided along this major alternative splicing ofreceptors whereby FGFRb-interacting FGFs are produced by epithelialcells and FGFRc-interacting FGFs are produced by mesenchymal cells(Ornitz et al., “Fibroblast Growth Factors,” Genome Biol2(3):reviews3005.1-reviews3005.12 (2001), which is hereby incorporatedby reference in its entirety). These reciprocal expression patterns ofFGFs and FGFRs result in the establishment of specific paracrine FGFsignaling loops between the epithelium and the mesenchyme, which isessential for proper organogenesis and patterning during embryonicdevelopment as well as tissue homeostasis in the adult organism.

Based on sequence homology and phylogenetic and structuralconsiderations, the eighteen mammalian FGFs are grouped into sixsubfamilies (Itoh et al., “Fibroblast growth factors: from molecularevolution to roles in development, metabolism, and disease,” J Biochem149:121-130 (2011); Mohammadi et al., “Structural basis for fibroblastgrowth factor receptor activation,” Cytokine Growth Factor Rev16:107-137 (2005), which are hereby incorporated by reference in itsentirety). The FGF core homology domain (approximately 120 amino acidslong) is flanked by N- and C-terminal sequences that are highly variablein both length and primary sequence, particularly among different FGFsubfamilies. The core region of FGF19 shares the highest sequenceidentity with FGF21 (38%) and FGF23 (36%), and therefore, these ligandsare considered to form a subfamily.

Based on mode of action, the eighteen mammalian FGFs are grouped intoparacrine-acting ligands (five FGF subfamilies) and endocrine-actingligands (one FGF subfamily) comprising FGF19, FGF21 and FGF23 (Itoh andOrnitz, “Fibroblast Growth Factors: From Molecular Evolution to Roles inDevelopment, Metabolism and Disease,” J. Biochem. 149:121-130 (2011);Mohammadi et al., “Structural Basis for Fibroblast Growth FactorReceptor Activation,” Cytokine Growth Factor Rev. 16:107-137 (2005),which are hereby incorporated by reference in their entirety).

Paracrine FGFs direct multiple processes during embryogenesis, includinggastrulation, somitogenesis, organogenesis, and tissue patterning (Itohand Ornitz, “Fibroblast Growth Factors: From Molecular Evolution toRoles in Development, Metabolism and Disease,” J. Biochem. 149:121-130(2011); Bottcher and Niehrs, “Fibroblast Growth Factor Signaling DuringEarly Vertebrate Development,” Endocr. Rev. 26:63-77 (2005); Thisse etal., “Functions and Regulations of Fibroblast Growth Factor SignalingDuring Embryonic Development,” Dev. Biol. 287:390-402 (2005), which arehereby incorporated by reference in their entirety), and also regulatetissue homeostasis in the adult (Hart et al., “Attenuation of FGFSignalling in Mouse Beta-cells Leads to Diabetes,” Nature 408:864-868(2000); Jonker et al., “A PPARγ-FGF1 Axis is Required for AdaptiveAdipose Remodelling and Metabolic Homeostasis,” Nature 485:391-394(2012), which is hereby incorporated by reference in its entirety).

Endocrine FGFs control major metabolic processes such as bile acidhomeostasis (Inagaki et al., “Fibroblast Growth Factor 15 Functions asan Enterohepatic Signal to Regulate Bile Acid Homeostasis,” Cell Metab.2:217-225 (2005), which is hereby incorporated by reference in itsentirety), and hepatic glucose and protein metabolism (Kir et al.,“FGF19 as a Postprandial, Insulin-Independent Activator of HepaticProtein and Glycogen Synthesis,” Science 331:1621-1624 (2011); Potthoffet al., “FGF15/19 Regulates Hepatic Glucose Metabolism by Inhibiting theCREB-PGC-1α Pathway,” Cell Metab. 13:729-738 (2011), which are herebyincorporated by reference in their entirety) (FGF19), glucose and lipidmetabolism (Badman et al., “Hepatic Fibroblast Growth Factor 21 IsRegulated by PPARα and Is a Key Mediator of Hepatic Lipid Metabolism inKetotic States,” Cell Metab. 5:426-437 (2007); Inagaki et al.,“Endocrine Regulation of the Fasting Response by PPARalpha-mediatedInduction of Fibroblast Growth Factor 21,” Cell Metab. 5:415-425 (2007);Kharitonenkov et al., “FGF-21 as a Novel Metabolic Regulator,” J. Clin.Invest. 115:1627-1635 (2005); Potthoff et al., “FGF21 Induces PGC-1alphaand Regulates Carbohydrate and Fatty Acid Metabolism During the AdaptiveStarvation Response,” Proc. Nat'l. Acad. Sci. U.S.A. 106:10853-10858(2009), which are hereby incorporated by reference in their entirety)(FGF21), and phosphate and vitamin D homeostasis (White et al.,“Autosomal Dominant Hypophosphataemic Rickets is Associated withMutations in FGF23,” Nat. Genet. 26:345-348 (2000); Shimada et al.,“Targeted Ablation of Fgf23 Demonstrates an Essential Physiological Roleof FGF23 in Phosphate and Vitamin D Metabolism,” J. Clin. Invest.113:561-568 (2004), which are hereby incorporated by reference in theirentirety) (FGF23). Thus, these ligands have attracted much attention aspotential drugs for the treatment of various inherited or acquiredmetabolic disorders (Beenken and Mohammadi, “The FGF Family: Biology,Pathophysiology and Therapy,” Nat. Rev. Drug Discov. 8:235-253 (2009);Beenken and Mohammadi, “The Structural Biology of the FGF19 Subfamily,”in Endocrine FGFs andKlothos (Kuro-o, M. ed.), Landes Bioscience. pp1-24 (2012), which are hereby incorporated by reference in theirentirety).

FGFs share a core homology region of about one hundred and twenty aminoacids that fold into a 3-trefoil (Ago et al., J. Biochem. 110:360-363(1991); Eriksson et al., Proc. Nat'l. Acad. Sci. U.S.A. 88:3441-3445(1991); Zhang et al., Proc. Nat'l. Acad. Sci. U.S.A. 88:3446-3450(1991); Zhu et al., Science 251:90-93 (1991), which are herebyincorporated by reference in their entirety) consisting of twelve βstrands in paracrine FGFs (β 1-β 12) and eleven β strands in endocrineFGFs (β 1-β 10 and β 12) (Mohammadi et al., “Structural Basis forFibroblast Growth Factor Receptor Activation,” Cytokine Growth FactorRev. 16:107-137 (2005); Goetz et al., Mol. Cell Biol. 27:3417-3428(2007), which are hereby incorporated by reference in their entirety).The conserved core region is flanked by divergent N- and C-termini,which play a critical role in conferring distinct biological activity onFGFs (Mohammadi et al., “Structural Basis for Fibroblast Growth FactorReceptor Activation,” Cytokine Growth Factor Rev. 16:107-137 (2005);Olsen et al., Genes Dev. 20:185-198 (2006), which are herebyincorporated by reference in their entirety).

All FGFs interact with pericellular heparan sulfate (HS)glycosaminoglycans albeit with different affinities (Asada et al.,Biochim. Biophys. Acta. 1790:40-48 (2009), which is hereby incorporatedby reference in its entirety). The HS-binding site of FGFs is comprisedof the β1-β2 loop and the region between β10 and β12 strands (Mohammadiet al., “Structural Basis for Fibroblast Growth Factor ReceptorActivation,” Cytokine Growth Factor Rev. 16:107-137 (2005), which ishereby incorporated by reference in its entirety). HS interacts withboth side chain and main chain atoms of the HS-binding site in paracrineFGFs (Schlessinger et al., Mol. Cell 6:743-750 (2000), which is herebyincorporated by reference in its entirety). The HS-binding site ofendocrine FGFs deviates from the common conformation adopted byparacrine FGFs such that interaction of HS with backbone atoms of theHS-binding site is precluded (Goetz et al., Mol. Cell Biol. 27:3417-3428(2007), which is hereby incorporated by reference in its entirety). As aresult, compared to paracrine FGFs, endocrine FGFs exhibit poor affinityfor HS (Beenken and Mohammadi, “The FGF Family: Biology, Pathophysiologyand Therapy,” Nat. Rev. Drug Discov. 8:235-253 (2009); Asada et al.,Biochim. Biophys. Acta. 1790:40-48 (2009), which are hereby incorporatedby reference in their entirety). The poor HS affinity enables theseligands to diffuse freely away from the site of their secretion andenter the blood circulation to reach their distant target organs (Goetzet al., Mol. Cell Biol. 27:3417-3428 (2007); Asada et al., Biochim.Biophys. Acta. 1790:40-48 (2009), which are hereby incorporated byreference in their entirety).

By contrast, owing to their high HS affinity (Asada et al., Biochim.Biophys. Acta. 1790:40-48 (2009), which is hereby incorporated byreference in its entirety), paracrine FGFs are mostly immobilized in thevicinity of the cells secreting these ligands, and hence can only actwithin the same organ. There is emerging evidence that differences inHS-binding affinity among paracrine FGFs translate into the formation ofligand-specific gradients in the pericellular matrix (Kalinina et al.,Mol. Cell Biol. 29:4663-4678 (2009); Makarenkova et al., Sci. Signal2:ra55 (2009), which are hereby incorporated by reference in theirentirety), which contribute to the distinct functions of these ligands(Beenken and Mohammadi, “The FGF Family: Biology, Pathophysiology andTherapy,” Nat. Rev. Drug Discov. 8:235-253 (2009); Itoh and Ornitz,“Fibroblast Growth Factors: From Molecular Evolution to Roles inDevelopment, Metabolism and Disease,” J. Biochem. 149:121-130 (2011),which are hereby incorporated by reference in their entirety).

Besides controlling ligand diffusion in the extracellular space, HSpromotes the formation of the 2:2 paracrine FGF-FGFR signal transductionunit (Schlessinger et al., Mol. Cell 6:743-750 (2000); Mohammadi et al.,Curr. Opin. Struct. Biol. 15:506-516 (2005), which are herebyincorporated by reference in their entirety). HS engages both ligand andreceptor to enhance the binding affinity of FGF for receptor and promotedimerization of ligand-bound receptors. Owing to their poor HS-bindingaffinity, endocrine FGFs rely on Klotho co-receptors to bind theircognate FGFR (Kurosu et al., J. Biol. Chem. 282:26687-26695 (2007);Kurosu et al., J. Biol. Chem. 281:6120-6123 (2006); Ogawa et al., Proc.Nat'l. Acad. Sci. U.S.A. 104:7432-7437 (2007); Urakawa et al., Nature444:770-774 (2006), which are hereby incorporated by reference in theirentirety). Klotho co-receptors are single-pass transmembrane proteinswith an extracellular domain composed of two type Iβ-glycosidase domains(Ito et al., Mech. Dev. 98:115-119 (2000); Kuro-o et al., Nature390:45-51 (1997), which are hereby incorporated by reference in theirentirety). Klotho co-receptors constitutively associate with FGFRs toenhance the binding affinity of endocrine FGFs for their cognate FGFRsin target tissues (Kurosu et al., J Biol. Chem. 282:26687-26695 (2007);Kurosu et al., J. Biol. Chem. 281:6120-6123 (2006); Ogawa et al., Proc.Nat'l. Acad. Sci. U.S.A. 104:7432-7437 (2007); Urakawa et al., Nature444:770-774 (2006), which are hereby incorporated by reference in theirentirety). αKlotho is the co-receptor for FGF23 (Kurosu et al., J. Biol.Chem. 281:6120-6123 (2006); Urakawa et al., Nature 444:770-774 (2006),which are hereby incorporated by reference in their entirety), andβKlotho is the co-receptor for both FGF19 and FGF21 (Kurosu et al., J.Biol. Chem. 282:26687-26695 (2007); Ogawa et al., Proc. Nat'l. Acad.Sci. U.S.A. 104:7432-7437 (2007), which are hereby incorporated byreference in their entirety). The C-terminal region of endocrine FGFsmediates binding of these ligands to the FGFR-α/βKlotho co-receptorcomplex (Goetz et al., Mol. Cell Biol. 27:3417-3428 (2007); Goetz etal., Proc. Nat'l. Acad. Sci. U.S.A 107:407-412 (2010); Micanovic et al.,J. Cell Physiol. 219:227-234 (2009); Wu et al., J. Biol. Chem.283:33304-33309 (2008); Yie et al., FEBS Lett, 583:19-24 (2009); Goetzet al., Mol. Cell Biol. 32:1944-1954 (2012), which are herebyincorporated by reference in their entirety).

βKlotho promotes binding of FGF21 to its cognate FGFR by engaging ligandand receptor simultaneously through two distinct binding sites (Goetz etal., “Klotho Coreceptors Inhibit Signaling by Paracrine FibroblastGrowth Factor 8 Subfamily Ligands,” Mol Cell Biol 32:1944-1954 (2012),which is hereby incorporated by reference in its entirety). βKlothoplays the same role in promoting binding of FGF19 to its cognate FGFR(Goetz et al., “Klotho Coreceptors Inhibit Signaling by ParacrineFibroblast Growth Factor 8 Subfamily Ligands,” Mol Cell Biol32:1944-1954 (2012), which is hereby incorporated by reference in itsentirety). The binding site for βKlotho was mapped on FGF21 and FGF19 tothe C-terminal region of each ligand that follows the β-trefoil coredomain (Goetz et al., “Klotho Coreceptors Inhibit Signaling by ParacrineFibroblast Growth Factor 8 Subfamily Ligands,” Mol Cell Biol32:1944-1954 (2012), which is hereby incorporated by reference in itsentirety). In the course of these studies, it was found that theC-terminal tail peptides of FGF21 and FGF19 share a common binding siteon βKlotho, and that the C-terminal tail of FGF19 binds tighter than theC-terminal tail of FGF21 to this site (Goetz et al., “Klotho CoreceptorsInhibit Signaling by Paracrine Fibroblast Growth Factor 8 SubfamilyLigands,” Mol Cell Biol 32:1944-1954 (2012), which is herebyincorporated by reference in its entirety).

Endocrine FGFs still possess residual HS-binding affinity, and moreover,there are differences in this residual binding affinity among theendocrine FGFs (Goetz et al., Mol. Cell Biol. 27:3417-3428 (2007), whichis hereby incorporated by reference in its entirety). These observationsraise the possibility that HS may play a role in endocrine FGFsignaling. Indeed, there are several reports showing that HS can promoteendocrine FGF signaling in the presence as well as in the absence ofKlotho co-receptor. It has been shown that HS augments the mitogenicsignal elicited by endocrine FGFs in BaF3 cells over-expressing FGFR andKlotho co-receptor by at least two-fold (Suzuki et al., Mol. Endocrinol.22:1006-1014 (2008), which is hereby incorporated by reference in itsentirety). In addition, even in the absence of Klotho co-receptor, HSenables endocrine FGFs to induce proliferation of BaF3 cellsover-expressing FGFR (Yu et al., Endocrinology 146:4647-4656 (2005);Zhang et al., J. Biol. Chem. 281:15694-15700 (2006), which are herebyincorporated by reference in their entirety). Compared to paracrineFGFs, however, significantly higher concentrations of both ligand and HSare needed, and the proliferative response of cells to endocrine FGFsstill lags behind that of paracrine FGFs by about one order of magnitude(Zhang et al., J. Biol. Chem. 281:15694-15700 (2006), which is herebyincorporated by reference in its entirety).

As used herein, the terms “chimeric polypeptide” and “chimeric protein”encompass a polypeptide having a sequence that includes at least aportion of a full-length sequence of first polypeptide sequence and atleast a portion of a full-length sequence of a second polypeptidesequence, where the first and second polypeptides are differentpolypeptides. A chimeric polypeptide also encompasses polypeptides thatinclude two or more non-contiguous portions derived from the samepolypeptide. A chimeric polypeptide or protein also encompassespolypeptides having at least one substitution, wherein the chimericpolypeptide includes a first polypeptide sequence in which a portion ofthe first polypeptide sequence has been substituted by a portion of asecond polypeptide sequence.

As used herein, the term “N-terminal portion” of a given polypeptidesequence is a contiguous stretch of amino acids of the given polypeptidesequence that begins at or near the N-terminal residue of the givenpolypeptide sequence. An N-terminal portion of the given polypeptide canbe defined by a contiguous stretch of amino acids (e.g., a number ofamino acid residues). Similarly, the term “C-terminal portion” of agiven polypeptide sequence is a contiguous length of the givenpolypeptide sequence that ends at or near the C-terminal residue of thegiven polypeptide sequence. A C-terminal portion of the givenpolypeptide can be defined by a contiguous stretch of amino acids (e.g.,a number of amino acid residues).

The term “portion,” when used herein with respect to a given polypeptidesequence, refers to a contiguous stretch of amino acids of the givenpolypeptide's sequence that is shorter than the given polypeptide'sfull-length sequence. A portion of a given polypeptide may be defined byits first position and its final position, in which the first and finalpositions each correspond to a position in the sequence of the givenfull-length polypeptide. The sequence position corresponding to thefirst position is situated N-terminal to the sequence positioncorresponding to the final position. The sequence of the portion is thecontiguous amino acid sequence or stretch of amino acids in the givenpolypeptide that begins at the sequence position corresponding to thefirst position and ending at the sequence position corresponding to thefinal position. A portion may also be defined by reference to a positionin the given polypeptide sequence and a length of residues relative tothe referenced position, whereby the sequence of the portion is acontiguous amino acid sequence in the given full-length polypeptide thathas the defined length and that is located in the given polypeptide inreference to the defined position.

As noted above, a chimeric protein according to the present inventionmay include an N-terminus coupled to a C-terminus. N-terminus andC-terminus are used herein to refer to the N-terminal region or portionand the C-terminal region or portion, respectively, of the chimericprotein of the present invention. In some embodiments of the presentinvention, the C-terminal portion and the N-terminal portion of thechimeric protein of the present invention are contiguously joined. Inalternative embodiments, the C-terminal portion and the N-terminalportion of the chimeric protein of the present invention are coupled byan intervening spacer. In one embodiment, the spacer may be apolypeptide sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more aminoacid residues. In some embodiments, the C-terminal portion and/or theN-terminal portion of the chimeric protein of the present invention mayinclude additional portion(s) coupled to the C-terminal residue and/orthe N-terminal residue of the chimeric protein of the present invention,respectively. In some embodiments, the additional portion(s) may be apolypeptide sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more aminoacid residues. In some embodiments, the N-terminal portion and/or theC-terminal portion having such additional portion(s) will maintain theactivity of the corresponding naturally occurring N-terminal portionand/or C-terminal portion, respectively. In some embodiments, theN-terminal portion and/or the C-terminal portion having such additionalportion(s) will have enhanced and/or prolonged activity compared to thecorresponding naturally occurring N-terminal portion and/or C-terminalportion, respectively. In other embodiments, the C-terminal portionand/or the N-terminal portion of the chimeric protein of the presentinvention do not include any additional portion(s) coupled to theC-terminal residue and/or the N-terminal residue of the chimeric proteinof the present invention, respectively.

The portion of the paracrine FGF may be derived from any suitableparacrine FGF. Suitable paracrine FGFs in accordance with the presentinvention include FGF1, FGF2, and ligands of the FGF4 and FGF9subfamilies. Certain embodiments of the present invention may include afull-length amino acid sequence of a paracrine FGF, rather than aportion of a paracrine FGF.

In one embodiment, the portion of the paracrine FGF is derived from amammalian FGF. In one embodiment, the portion of the paracrine FGF isderived from a vertebrate FGF. In one embodiment, the portion of theparacrine FGF is derived from a human FGF. In one embodiment, theparacrine FGF is derived from a non-human mammalian FGF. In oneembodiment, the portion of the paracrine FGF is derived from a non-humanvertebrate FGF. In one embodiment, the paracrine FGF is derived from anortholog of human FGF, or a polypeptide or protein obtained from onespecies that is the functional counterpart of a polypeptide or proteinfrom a different species.

In one embodiment according to the present invention, the portion of theparacrine FGF of the chimeric protein includes an N-terminal portion ofthe paracrine FGF.

In one embodiment, the paracrine FGF is FGF1. In one embodiment, theportion of the FGF1 is from human FGF1 having the following amino acidsequence (GenBank Accession No. AAH32697, which is hereby incorporatedby reference in its entirety) (SEQ ID NO: 1):

1 MAEGEITTFT ALTEKFNLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ 61LSAESVGEVY IKSTETGQYL AMDTDGLLYG SQTPNEECLF LERLEENHYN TYISKKHAEK 121NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD

In one embodiment, the portion of the paracrine FGF includes an aminoacid sequence beginning at any one of residues 1 to 25 and ending at anyone of residues 150 to 155 of SEQ ID NO: 1 (human FGF1). In oneembodiment, the portion of the paracrine FGF includes amino acidresidues 1-150, 1-151, 1-152, 1-153, 1-154, 1-155, 2-150, 2-151, 2-152,2-153, 2-154, 2-155, 3-150, 3-151, 3-152, 3-153, 3-154, 3-155, 4-150,4-151, 4-152, 4-153, 4-154, 4-155, 5-150, 5-151, 5-152, 5-153, 5-154,5-155, 6-150, 6-151, 6-152, 6-153, 6-154, 6-155, 7-150, 7-151, 7-152,7-153, 7-154, 7-155, 8-150, 8-151, 8-152, 8-153, 8-154, 8-155, 9-150,9-151, 9-152, 9-153, 9-154, 9-155, 10-150, 10-151, 10-152, 10-153,10-154, 10-155, 11-150, 11-151, 11-152, 11-153, 11-154, 11-155, 12-150,12-151, 12-152, 12-153, 12-154, 12-155, 13-150, 13-151, 13-152, 13-153,13-154, 13-155, 14-150, 14-151, 14-152, 14-153, 14-154, 14-155, 15-150,15-151, 15-152, 15-153, 15-154, 15-155, 16-150, 16-151, 16-152, 16-153,16-154, 16-155, 17-150, 17-151, 17-152, 17-153, 17-154, 17-155, 18-150,18-151, 18-152, 18-153, 18-154, 18-155, 19-150, 19-151, 19-152, 19-153,19-154, 19-155, 20-150, 20-151, 20-152, 20-153, 20-154, 20-155, 21-150,21-151, 21-152, 21-153, 21-154, 21-155, 22-150, 22-151, 22-152, 22-153,22-154, 22-155, 23-150, 23-151, 23-152, 23-153, 23-154, 23-155, 24-150,24-151, 24-152, 24-153, 24-154, 24-155, 25-150, 25-151, 25-152, 25-153,25-154, or 25-155 of FGF1 (SEQ ID NO: 1). In one embodiment, the portionof the paracrine FGF includes amino acid residues 1-150 or 25-150 of SEQID NO: 1.

In one embodiment, the portion of the paracrine FGF includes an aminoacid sequence that has at least 80%, at least 85%, at least 90%, atleast 95%, at least 97% or at least 99% amino acid sequence identity toan amino acid sequence beginning at any one of residues 1 to 25 andending at any one of residues 150 to 155 of SEQ ID NO: 1 (human FGF1).In one embodiment, the portion of the paracrine FGF includes an aminoacid sequence that has at least 80%, at least 85%, at least 90%, atleast 95%, at least 97% or at least 99% amino acid sequence homology toan amino acid sequence beginning at any one of residues 1 to 25 andending at any one of residues 150 to 155 of SEQ ID NO: 1 (human FGF1).

Percent (%) amino acid sequence identity with respect to a givenpolypeptide sequence identified herein is defined as the percentage ofamino acid residues in a candidate sequence that are identical to theamino acid residues in the reference sequence, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. Percent (%) amino acidsequence homology with respect to a given polypeptide sequenceidentified herein is the percentage of amino acid residues in acandidate sequence that are identical to or strongly similar to theamino acid residues in the reference sequence, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence homology. Strongly similar amino acid residues mayinclude, for example, conservative amino acid substitutions known in theart. Alignment for purposes of determining percent amino acid sequenceidentity and/or homology can be achieved in various ways that are withinthe skill in the art, for instance, using publicly available computersoftware such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor measuring alignment, including any algorithms needed to achievemaximal alignment over the full-length of the sequences being compared.

In one embodiment of the present invention, the portion of the paracrineFGF of the chimeric protein is derived from an ortholog of human FGF1.In one embodiment, the portion of FGF1 is derived from Papio Anubis,Pongo abelii, Callithrix jacchus, Equus caballus, Pan troglodytes,Loxodonta Africana, Canis lupus familiaris, Ailuropoda melanoleuca,Saimiri boliviensis boliviensis, Sus scrofa, Otolemur garnettii,Rhinolophus ferrumequinum, Sorex araneus, Oryctolagus cuniculus,Cricetulus griseus, Sarcophilus harrisii, Mus musculus, Cavia porcellus,Monodelphis domestica, Desmodus rotundus, Bos taurus, Ornithorhynchusanatinus, Taeniopygia guttata, Dasypus novemcinctus, Xenopus Siluranatropicalis, Heterocephalus glaber, Pteropus alecto, Tupaia chinensis,Columba livia, Ovis aries, Gallus gallus, Vicugna pacos, Anoliscarolinensis, Otolemur garnettii, Felis catus, Pelodiscus sinensis,Latimeria chalumnae, Tursiops truncates, Mustela putorius furo, Nomascusleucogenys, Gorilla gorilla, Erinaceus europaeus, Procavia capensis,Dipodomys ordii, Petromyzon marinus, Echinops telfairi, Macaca mulatta,Pteropus vampyrus, Myotis lucifugus, Microcebus murinus, Ochotonaprinceps, Rattus norvegicus, Choloepus hoffmanni, Ictidomystridecemlineatus, Tarsius syrichta, Tupaia belangeri, Meleagrisgallopavo, Macropus eugenii, or Danio rerio. The portions of an orthologof human paracrine FGF1 include portions corresponding to theabove-identified amino acid sequences of human FGF1. Correspondingportions may be determined by, for example, sequence analysis andstructural analysis.

In one embodiment, the portion of the FGF1 of the chimeric protein ofthe present invention is derived from an ortholog of human FGF1 havingthe amino acid sequence shown in Table 1.

TABLE 1 Amino acid sequence of human FGF1 (SEQ ID NO: 1)(GenBankaccession no. AAH32697, which is hereby incorporated by reference in itsentirety): 1 MAEGEITTFT ALTEKFNLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTRDRSDQHIQLQ 61 LSAESVGEVY IKSTETGQYL AMDTDGLLYG SQTPNEECLF LERLEENHYNTYISKKHAEK 121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD Amino acidsequence of Papio anubis (olive baboon) FGF1(SEQ ID NO: 2) (GenBankaccession no. NP_001162557, which is hereby incorporated by reference inits entirety): 1 MAEGEITTFT ALTEKFNLPP ANYKKPKLLY CSNGGHFLRI LPDGTVDGTRDRSDQHIQLQ 61 LSAESVGEVY IKSTETGQYL AMDTDGLLYG SQTPNEECLF LERLEENHYNTYISKKHAEK 121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD Amino acidsequence of Pongo abelii (Sumatran orangutan) FGF1(SEQ ID NO: 3)(GenBank accession no. NP_001127073, which is hereby incorporated byreference in its entirety) 60                                                                M 61AEGEITTFTA LTEKFNLPPG NYKKPKLLYC SNGGHFLRIL PDGTVDGTRD RSDQHIQLQL 121SAESVGEVYI KSTETGQYLA MDTDGLLYGS QTPNEECLFL ERLEENHYNT YISKKHAEKN 181WFVGLKKNGS CKRGPRTHYG QKAILFLPLP VSSD Amino acid sequence of Callithrixjacchus (white-tufted-ear marmoset) FGF1(SEQ ID NO: 4) (GenBankaccession no. XP_002744341, which is hereby incorporated by reference inits entirety): 1 MAEGEITTFT ALTEKFDLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTRDRSDQHIQLQ 61 LSAESVGEVY IKSTETGQYL AMDTDGLLYG SQTPNEECLF LERLEENHYNTYISKKHAEK 121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD Amino acidsequence of Equus caballus (horse) FGF1(SEQ ID NO: 5) (GenBank accessionno. NP_001157358, which is hereby incorporated by reference in itsentirety): 1 MAEGEITTFT ALTEKFNLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTRDRSDQHIQLQ 61 LSAESVGEVY IKSTETGQYL AMDTDGLLYG SQTPNEECLF LERLEENHYNTYTSKKHAEK 121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD Amino acidsequence of Pan troglodytes (chimpanzee) FGF1(SEQ ID NO: 6) (GenBankaccession no. JAA29511, which is hereby incorporated by reference in itsentirety): 1 MAEGEITTFT ALTEKFNLPS GNYKKPKLLY CSNGGHFLRI LPDGTVDGTRDRSDQHIQLQ 61 LSAESVGEVY IKSTETGQYL AMDTDGLLYG SQTPNEECLF LERLEENHYNTYISKKHAEK 121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD Amino acidsequence of Loxodonta africana (elephant) FGF1(SEQ ID NO: 7) (GenBankaccession no. XP_003404621, which is hereby incorporated by reference inits entirety): 1 MAEGEITTFT ALTEKFNLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTRDRSDQHIQLQ 61 LSAESVGEVY IKGTETGQYL AMDTDGLLYG SQTPNEECLF LERLEENHYNTYTSKKHAEK 121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD Amino acidsequence of Canis lupus familiaris (dog) FGF1(SEQ ID NO: 8) (GenBankaccession no. XP_849274, which is hereby incorporated by reference inits entirety): 1 MAEGEITTFT ALTEKFNLPP GNYMKPKLLY CSNGGHFLRI LPDGTVDGTRDRSDQHIQLQ 61 LSAESVGEVY IKSTETGQYL AMDTDGLLYG SQTPNEECLF LERLEENHYNTYTSKKHAEK 121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD Amino acidsequence of Ailuropoda melanoleuca (giant panda) FGF1(SEQ ID NO: 9)(GenBank accession no. XP_002912581, which is hereby incorporated byreference in its entirety): 1 MAEGEITTFT ALTEKFNLPA GNYKKPKLLYCSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ 61 LSAESVGEVY IKSTETGQYL AMDTDGLLYGSQTPNEECLF LERLEENHYN TYTSKKHAEK 121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPLPVSSD Amino acid sequence of Saimiri boliviensis boliviensis (Boliviansquirrel monkey) FGF1(SEQ ID NO: 10) (GenBank accession no.XP_003920596, which is hereby incorporated by reference in itsentirety): 1 MAEGEITTFT ALTEKFDLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTRDRSDLHIQLQ 61 LSAESVGEVY IKSTETGQYL AMDTDGLLYG SQTPNEECLF LERLEENHYNTYISKKHAEK 121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD Amino acidsequence of Sus scrofa (pig) FGF1(SEQ ID NO: 11) (GenBank accession no.XP_003124058, which is hereby incorporated by reference in itsentirety): 1 MAEGEITTFT ALTEKFNLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTRDRSDQHIQLQ 61 LSAESVGEVY IKSTETGQYL AMDTSGLLYG SQTPSEECLF LERLEENHYNTYTSKKHAEK 121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD Amino acidsequence of Otolemur garnettii (small-eared galago) FGF1(SEQ ID NO: 12)(GenBank accession no. XP_003782135, which is hereby incorporated byreference in its entirety): 1 MAEGEITTFT ALTEKFNLPL GNYKKPKLLYCSNGGHFLRI LPDGTVDGTQ DRSDQHIQLQ 61 LSAESVGEVY IKSTQTGQYL AMDSDGLLYGSQTPNEECLF LERLEENHYN TYVSKKHAEK 121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPLPVSSD Amino acid sequence of Rhinolophus ferrumequinum (greaterhorseshoe bat) FGF1(SEQ ID NO: 13) (GenBank accession no. ACC62496,which is hereby incorporated by reference in its entirety): 1 MAEGEVTTFTALTEKFNLPT GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DKSDQHIQLQ 61 LSAESVGEVYIKSTESGQYL AMDSDGLLYG SQTPNEECLF LERLEENHYN TYTSKKHAEK 121 NWFVGLKKNGSCKRGPRTHY GQKAILFLPL PVSSD Amino acid sequence of Sorex araneus(European shrew) FGF1(SEQ ID NO: 14) (GenBank accession no. ACE75805,which is hereby incorporated by reference in its entirety): 1 MAEGEITTFGALMEKFNLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ 61 LSAESVGEVYIKSTETGHYL AMDTDGLLYG SQTPNEECLF LERLEENHYN TYTSKKHAEK 121 NWFVGLKKNGSCKRGPRTHY GQKAILFLPL PVSSD Amino acid sequence of Oryctolagus cuniculus(rabbit) FGF1(SEQ ID NO: 15) (GenBank accession no. NP_001164959, whichis hereby incorporated by reference in its entirety): 1 MAEGEVTTFTALTEKFNLPA GNYKLPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ 61 LSAESVGEVYIKSTETGQYL AMDTDGLLYG SQTPSEECLF LERLEENHYN TYTSKKHAEK 121 NWFVGLKKNGSCKRGPRTHY GQKAILFLPL PVSSD Amino acid sequence of Cricetulus griseus(Chinese hamster) FGF1(SEQ ID NO: 16) (GenBank accession no.XP_003502469, which is hereby incorporated by reference in itsentirety): 1 MAEGEITTFS ALTERFNLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTRDRSDQHIQLQ 61 LSAESAGEVY IKGTETGQYR NMDTDGLLYG SQTPNEECLF LERLEENHYNTYTSKKHAEK 121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD Amino acidsequence of Sarcophilus harrisii (Tasmanian devil) FGF1(SEQ ID NO: 17)(GenBank accession no. XP_003756738, which is hereby incorporated byreference in its entirety): 1 MAEGEITTFT ALTERFNLPL GNYKKPKLLYCSNGGHFLRI LPDGKVDGTR DRNDQHIQLQ 61 LSAESVGEVY IKSTESGQYL AMDTDGLLYGSQTPTEECLF LERLEENHYN TYISKKHAEK 121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPLPVSSE Amino acid sequence of Mus musculus (house mouse) FGF1(SEQ ID NO:18) (GenBank accession no. NP_034327, which is hereby incorporated byreference in its entirety): 1 MAEGEITTFA ALTERFNLPL GNYKKPKLLYCSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ 61 LSAESAGEVY IKGTETGQYL AMDTEGLLYGSQTPNEECLF LERLEENHYN TYTSKKHAEK 121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPLPVSSD Amino acid sequence of Cavia porcellus (domestic guinea pig)FGF1(SEQ ID NO: 19) (GenBank accession no. XP_003477242, which is herebyincorporated by reference in its entirety): 1 MAEGEITTFA ALTEKFNLPPGNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ 61 LSAEGVGEVY IQSTETGQYLAMDTDGLLYG SQTPSEECLF LERLEENHYN TYTSKKHVEK 121 NWFVGLKKNG SCKRGPRTHYGQKAILFLPL PVSD Amino acid sequence of Monodelphis domestica (grayshort-tailed opossum) FGF1(SEQ ID NO: 20) (GenBank accession no.XP_001368921, which is hereby incorporated by reference in itsentirety): 1 MAEGEITTFT ALTERFNLPL GNYKKPKLLY CSNGGHFLRI LPDGKVDGTRDRNDQHIQLQ 61 LSTESVGEVY IKSTESGQYL AMDTDGLLYG SQTPSEECLF LERLEENHYNTYTSKKHAEK 121 NWFVGLKKNG SCKKGPRTHY GQKAILFLPL PVSSE Amino acidsequence of Desmodus rotundus (common vampire bat) FGF1(SEQ ID NO: 21)(GenBank accession no. JAA45191, which is hereby incorporated byreference in its entirety): 1 MAEGEVTTFT ALTEKFNLPL ESYKKPKLLYCSNGGHFLRI LPDGTVDGTR DKSDQHIQLQ 61 LSAESVGEVY IKSTGSGQYL AMDSAGLLYGSQTPNEECLF LERLEENHYN TYTSKKHAEK 121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPLPVNSD Amino acid sequence of Bos taurus (cattle) FGF1(SEQ ID NO: 22)(GenBank accession no. NP_776480, which is hereby incorporated byreference in its entirety): 1 MAEGETTTFT ALTEKFNLPL GNYKKPKLLYCSNGGYFLRI LPDGTVDGTK DRSDQHIQLQ 61 LCAESIGEVY IKSTETGQFL AMDTDGLLYGSQTPNEECLF LERLEENHYN TYISKKHAEK 121 HWFVGLKKNG RSKLGPRTHF GQKAILFLPLPVSSD Amino acid sequence of Ornithorhynchus anatinus (platypus)FGF1(SEQ ID NO: 23) (GenBank accession no. XP_001514861, which is herebyincorporated by reference in its entirety): 1 MAEGEITTFT ALMEKFDLPLGNYKKPRLLY CSNGGYFLRI QPDGKVDGTR DRSDQHIQLQ 61 LSAESVGEVY IKSTESGHYLAMDTEGLLYG SQAPSEDCLF LERLEENHYN TYVSKKHAEK 121 NWFVGLKKNG SCKRGPRTHYGQKAILFLPL PVASD Amino acid sequence of Taeniopygia guttata (zebrafinch) FGF1(SEQ ID NO: 24) (GenBank accession no. XP_002193287, which ishereby incorporated by reference in its entirety): 1 MAEGEITTFSALTEKFNLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ 61 LSAESVGVVHIQSTQSGQYL AMDTNGLLYG SQLPPGECLF LERLEENHYN TYVSKMHADK 121 NWFVGLKKNGTSKLGPRTHY GQKAILFLPL PVAAD Amino acid sequence of Dasypus novemcinctus(nine-banded armadillo) FGF1(SEQ ID NO: 25) (GenBank accession no.ACO06224, which is hereby incorporated by reference in its entirety): 1MAEGEITTFM ALMEKFNLPL ENYKHPRLLY CRNGGHFLRI LPDGTVDGTR DRSDQHIQLQ 61LSAESVGEVY IKSAETGQYL AMDTDGLLYG SETPSEECLF MEKLEENNYN TYISKKHAEK 121KWFVGLKKDG SSKRGPQTHY GQKAILFLPL PVSSD Amino acid sequence of XenopusSilurana tropicalis (western clawed frog) FGF1(SEQ ID NO: 26) (GenBankaccession no. ACJ50585, which is hereby incorporated by reference in itsentirety): 1 MAEGDITTFN PIAESFSLPI GNYKKPKLLY CNNGGYFLRI LPDGVVDGTRDRDDLYITLK 61 LSAQSQGEVH IKSTETGSYL AMDSSGQLYG TLTPNEESLF LETLEENHYNTYKSKKYAEN 121 NWFVGIKKNG ASKKGSRTHY GQKAILFLPL PASPD Amino acidsequence of Heterocephalus glaber (naked mole-rat) FGF1(SEQ ID NO: 27)(GenBank accession no. EHA99379, which is hereby incorporated byreference in its entirety): 1 MAEGEITTFT ALTEKFNLPP GNYKKPKLLYCSNGGHFLRI LPDGKVDGTR DRSDQHIQLQ 61 LSAEGVGEVY IKSTETGQYL AMDTDGLLYGSQTASEECLF LERLEENHYN TYISKKHAEK 121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPLPVSSD Amino acid sequence of Pteropus alecto (black flying fox) FGF1(SEQID NO: 28) (GenBank accession no. ELK02961, which is hereby incorporatedby reference in its entirety): 1 MAEGEVTTFT ALTERFNLPP GNYKKPKLLYCSNGGHFLRI LPDGTVDGTR DKSDQHIQLQ 61 LSAESVGEVY IKSTESGQYL AMDSDGLLYGSQTPDEDCLF LERLEENHYN TYTSKKHAEK 121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPLPVSSD Amino acid sequence of Tupaia chinensis (Chinese tree shrew)FGF1(SEQ ID NO: 29) (GenBank accession no. ELW69091, which is herebyincorporated by reference in its entirety): 1 MAEGEITTFA ALTEKFDLPPGNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ 61 LTAENVGEVY IKSTETGQYLAMDADGLLYG SQTPNEECLF LERLEENHYN TYISKKHAEK 121 NWFVALKKNG SCKLGPRTHYGQKAILFLPL PVSSD Amino acid sequence of Columba livia (rock pigeon)FGF1(SEQ ID NO: 30) (GenBank accession no. EMC79997, which is herebyincorporated by reference in its entirety): 1 MAEGEITTFT ALTEKFNLPPGNYKKPKLLY CSNGGHFLRI LPDGKVDGTR DRSDQHIQLQ 61 LSAESVGEVY IKSTQSGQYLAMDPTGLLYG SQLLGEECLF LERIEENHYN TYVSKKHADK 121 NWFVGLKKNG NSKLGPRTHYGQKAILFLPL PVSAD Amino acid sequence of Ovis aries (sheep) FGF1(SEQ IDNO: 31) (GenBank accession no. XP_004008958, which is herebyincorporated by reference in its entirety): 1 MAEGETTTFR ALTEKFNLPLGNYKKPKLLY CSNGGYFLRI LPDGRVDGTK DRSDQHIQLQ 61 LYAESIGEVY IKSTETGQFLAMDTNGLLYG SQTPSEECLF LERLEENHYN TYISKKHAEK 121 NWFIGLKKNG SSKLGPRTHFGQKAILFLPL PVSSD Amino acid sequence of Gallus gallus (chicken) FGF1(SEQID NO: 32) (GenBank accession no. NP_990511, which is herebyincorporated by reference in its entirety): 1 MAEGEITTFT ALTERFGLPLGNYKKPKLLY CSNGGHFLRI LPDGKVDGTR DRSDQHIQLQ 61 LSAEDVGEVY IKSTASGQYLAMDTNGLLYG SQLPGEECLF LERLEENHYN TYISKKHADK 121 NWFVGLKKNG NSKLGPRTHYGQKAILFLPL PVSAD Amino acid sequence of Vicugna pacos (alpaca) FGF1(SEQID NO: 33) (Ensembl accession no. ENSVPAP00000007810; partial sequencecorresponding to human FGF1 residues 58 to 155, which is herebyincorporated by reference in its entirety): 1 QLQLSAESVG EVYIKSTETGQYLAMDTDGL LHGSQTPNEE CLFLERLEEN HYNTYTSKKH 61 AEKNWFVGLK KNGSCKRGPRTHYGQKAILF LPLPVSSD Amino acid sequence of Anolis carolinensis (anolelizard) FGF1(SEQ ID NO: 34) (Ensembl accession no. ENSACAP00000013203,which is hereby incorporated by reference in its entirety): 1 MAEGEITTFTALTERFALPM ENYKKPKLLY CSNGGHFLRI LPDGKVDGTM DRNDSYIQLL 61 LTAEDVGVVYIKGTETGQYL AMDANGHLYG SQLPTEECLF VETLEENHYN TYTSKMHGDK 121 KWYVGLKKNGKGKLGPRTHR GQKAILFLPL PVSPD Amino acid sequence of Otolemur garnettii(bushbaby) FGF1(SEQ ID NO: 35) (Ensembl accession no.ENSOGAP00000004540, which is hereby incorporated by reference in itsentirety): 1 MAEGEITTFT ALTEKFNLPL GNYKKPKLLY CSNGGHFLRI LPDGTVDGTQDRSDQHIQLQ 61 LSAESVGEVY IKSTQTGQYL AMDSDGLLYG SQTPNEECLF LERLEENHYNTYVSKKHAEK 121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD Amino acidsequence of Felis catus (cat) FGF1(SEQ ID NO: 36) (Ensembl accession no.ENSFCAP00000008457, which is hereby incorporated by reference in itsentirety): 1 MAEGEITTFT ALTEKFNLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTRDRSDQHIQLQ 61 LSAESVGEVY IKSTETGQYL AMDTDGLLYG SQTPNEECLF LERLEENHYNTYTSKKHAEK 121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD Amino acidsequence of Pelodiscus sinensis (Chinese softshell turtle) FGF1(SEQ IDNO: 37) (Ensembl accession no. ENSPSIP00000016356, which is herebyincorporated by reference in its entirety): 1 MAEGEITTFT ALTEKFNLPLGNYKNPKLLY CSNGGYFLRI HPDGKVDGTR DRSDQHIQLQ 61 LSAESVGEVY IKSTESGQFLAMDANGLLYG SLSPSEECLF LERMEENHYN TYISKKHADK 121 NWFVGLKKNG SCKLGPRTHYGQKAVLFLPL PVSAD Amino acid sequence of Latimeria chalumnae (coelacanth)FGF1(SEQ ID NO: 38) (Ensembl accession no. ENSLACP00000015106, which ishereby incorporated by reference in its entirety): 1 MAEDKITTLKALAEKFNLPM GNYKKAKLLY CSNGGYFLRI PPDGKVEGIR ERSDKYIQLQ 61 MNAESLGMVSIKGVEAGQYL AMNTNGLLYG SQSLTEECLF MEKMEENHYN TYRSKTHADK 121 NWYVGIRKNGSIKPGPRTHI GQKAVLFLPL PASSD Amino acid sequence of Tursiops truncatus(dolphin) FGF1(SEQ ID NO: 39) (Ensembl accession no. ENSTTRP00000004470,which is hereby incorporated by reference in its entirety): 1 MAEGEITTFTALTEKFNLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ 61 LSAESVGEVYIKSTETGQYL AMDTDGLLYG SQTPNEECLF LERLEENHYN TYASKKHAEK 121 NWFVGLKKNGSCKRGPRTHY GQKAILFLPL PVSSD Amino acid sequence of Mustela putorius furo(ferret) FGF1(SEQ ID NO: 40) (Ensembl accession no. ENSMPUP00000007888,which is hereby incorporated by reference in its entirety): 1 MAEGEITTFTALMEKFNLPA GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ 61 LSAESVGEVYIKSTETGQYL AMDTDGLLYG SQTPNEECLF LERLEENHYN TYTSKKHAEK 121 NWFVGLKKNGSCKRGPRTHY GQKAILFLPL PVSSD Amino acid sequence of Nomascus leucogenys(gibbon) FGF1(SEQ ID NO: 41) (Ensembl accession no. ENSNLEP00000011873,which is hereby incorporated by reference in its entirety): 1 MAEGEITTFTALTEKFNLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ 61 LSAESVGEVYIKSTETGQYL AMDTDGLLYG SQTPNEECLF LERLEENHYN TYISKKHAEK 121 NWFVGLKKNGSCKRGPRTHY GQKAILFLPL PVSSD Amino acid sequence of Gorilla gorilla(gorilla) FGF1(SEQ ID NO: 42) (Ensembl accession no. ENSGGOP00000017663,which is hereby incorporated by reference in its entirety): 1 MAEGEITTFTALTEKFNLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ 61 LSAESVGEVYIKSTETGQYL AMDTDGLLYG SQTPNEECLF LERLEENHYN TYISKKHAEK 121 NWFVGLKKNGSCKRGPRTHY GQKAILFLPL PVSSD Amino acid sequence of Erinaceus europaeus(hedgehog) FGF1(SEQ ID NO: 43) (Ensembl accession no.ENSEEUP00000005318, which is hereby incorporated by reference in itsentirety): 1 MAEGEITTFT ALTEKFNLPL GNYKKPKLLY CSNGGHFLRI LPDGTVDGTRDRSDQHIQLQ 61 LSAESVGEVY IKSTETGQYL AMDTDGLLYG SQTPNEECLF LERLEENHYNTYTSKKHAEK 121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD Amino acidsequence of Procavia capensis (hyrax) FGF1(SEQ ID NO: 44) (Ensemblaccession no. ENSPCAP00000010969, which is hereby incorporated byreference in its entirety)(partial sequence corresponding to human FGF1residues 1 to 91): 1 MAEGEITTFT ALTEKFNLPL ENYKKPKLLY CSNGGHFLRILPDGTVDGTR DRSDQHIQLQ 61 LSAESVGEVY IKGTETGQYL AMDTDGLLYG S Amino acidsequence of Dipodomys ordii (kangaroo rat) FGF1(SEQ ID NO: 45) (Ensemblaccession no. ENSDORP00000006889, which is hereby incorporated byreference in its entirety) (partial sequence corresponding to human FGF1residues 1 to 16 and 58 to 155): 1 MAEGEITTFT ALTERF---- -------------------- ---------- -------QLQ 61 LSAESVGEVY IKSTETGQYL AMDADGLLYGSQTPDEECLF LERLEENHYN TYIAKKHAEK 121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPLPVSSD Amino acid sequence of Petromyzon marinus (lamprey) FGF1(SEQ IDNO: 46) (Ensembl accession no. ENSPMAP00000010683, which is herebyincorporated by reference in its entirety)(partial sequencecorresponding to human FGF1 residues 1 to 93): 1 MEVGHIGTLP VVPAGPVFPGSFKEPRRLYC RSAGHHLQIL GDGTVSGTQD ENEPHAVLQL 61 QAVRRGVVTI RGLCAERFLAMSTEGHLYGA VR Amino acid sequence of Echinops telfairi (lesser hedgehogtenrec) FGF1(SEQ ID NO: 47) (Ensembl accession no. ENSETEP00000014504,which is hereby incorporated by reference in its entirety)(partialsequence corresponding to human FGF1 residues 58 to 155) 1 QLKLVAESVGVVYIKSIKTG QYLAMNPDGL LYGSETPEEE CLFLETLEEN HYTTFKSKKH 61 VEKNWFVGLRKNGRVKIGPR THQGQKAILF LPLPVSSD Amino acid sequence of Macaca mulatta(rhesus monkey) FGF1(SEQ ID NO: 48) (Ensembl accession no.ENSMMUP00000030943, which is hereby incorporated by reference in itsentirety): 1 MAEGEITTFT ALTEKFNLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTRDRSDQHIQLQ 61 LSAESVGEVY IKSTETGQYL AMDTDGLLYG SQTPNEECLF LERLEENHYNTYTSKKHAEK 121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD Amino acidsequence of Pteropus vampyrus (megabat) FGF1(SEQ ID NO: 49) (Ensemblaccession no. ENSPVAP00000004349, which is hereby incorporated byreference in its entirety): 1 MAEGEVTTFT ALTERFNLPP GNYKKPKLLYCSNGGHFLRI LPDGTVDGTR DKSDQHIQLQ 61 LSAESVGEVY IKSTESGQYL AMDSDGLLYGSQTPDEDCLF LERLEENHYN TYTSKKHAEK 121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPLPVSSD Amino acid sequence of Myotis lucifugus (microbat) FGF1(SEQ ID NO:50) (Ensembl accession no. ENSMLUP00000006481, which is herebyincorporated by reference in its entirety): 1 MAEGEVTTFT ALTERFNLPLENYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ 61 LSAESVGEVY IKSTESGQYLAMDSDGLLYG SQTPNEECLF LERLEENHYN TYTSKKHAEK 121 NWFVGLKKNG SCKRGPRTHYGQKAILFLPL PVSSD Amino acid sequence of Microcebus murinus (mouse lemur)FGF1(SEQ ID NO: 51) (Ensembl accession no. ENSMICP00000008602, which ishereby incorporated by reference in its entirety): 1 MAEGEITTFTALTEKFNLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ 61 LSAESAGEVYIKSTQTGRYL AMDADGLLYG SQTPNEECLF LERLEENHYN TYVSKKHAEK 121 NWFVGLKKNGSCKRGPRTHY GQKAILFLPL PVSSD Amino acid sequence of Ochotona princeps(pika) FGF1(SEQ ID NO: 52) (Ensembl accession no. ENSOPRP00000011739,which is hereby incorporated by reference in its entirety): 1 MAEGEVTTFSALTEKFNLPG GNYKLPKLLY CSNGGHFLRI LPDGTVDGTR DRSDLH---- 61 -------EVFIKSTETGQYL AMDTDGLLYG SQTPSEECLF LERLEENHYN TYTSKKHAEK 121 NWFVGIKKNGSCKRGPRTHY GQKAILFLPL PVSSD Amino acid sequence of Rattus norvegicus(rat) FGF1(SEQ ID NO: 53) (Ensembl accession no. ENSRNOP00000018577,which is hereby incorporated by reference in its entirety): 1 MAEGEITTFAALTERFNLPL GNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ 61 LSAESAGEVYIKGTETGQYL AMDTEGLLYG SQTPNEECLF LERLEENHYN TYTSKKHAEK 121 NWFVGLKKNGSCKRGPRTHY GQKAILFLPL PVSSD Amino acid sequence of Choloepus hoffmanni(sloth) FGF1(SEQ ID NO: 54) (Ensembl accession no. ENSCHOP00000010964,which is hereby incorporated by reference in its entirety): 1 MAEGEITTFTALMEKFNLPP GNYMKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDLHIQLQ 61 LSAESVGEVYIKSAETGQYL AMDTGGLLYG SQTPSEECLF LERLEENHYN TYVSKKHAEK 121 NWFVGLKKNGSSKRGPRTHY GQKAILFLPL PVSSD Amino acid sequence of Ictidomystridecemlineatus (squirrel) FGF1(SEQ ID NO: 55) (Ensembl accession no.ENSSTOP00000021782, which is hereby incorporated by reference in itsentirety): 1 MAEGEITTFT ALTEKFNLPP GNYKKPKLLY CSNGGHFLRI LPDGTVDGTRDRSDQHIQLQ 61 LSAESVGEVY IKSTETGQYL AMDTDGLLYG SQTPNEECLF LERLEENHYNTYTSKKHAEK 121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD Amino acidsequence of Tarsius syrichta (tarsier) FGF1(SEQ ID NO: 56) (Ensemblaccession no. ENSTSYP00000006804, which is hereby incorporated byreference in its entirety): 1 MAEGEITTFT ALTEKFNLPP GNYKKPKLLYCSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ 61 LSAESVGEVY IKSTETGQYL AMDTDGLLYGSQTPNEECLF LERLEENHYN TYVSKKHAEK 121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPLPVSSD Amino acid sequence of Tupaia belangeri (tree shrew) FGF1(SEQ IDNO: 57) (Ensembl accession no. ENSTBEP00000010264, which is herebyincorporated by reference in its entirety): 1 MAEGEITTFA ALTEKFDLPPGNYKKPKLLY CSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ 61 LTAENVGEVY IKSTETGQYLAMDADGLLYG SQTPNEECLF LERLEENHYN TYISKKHAEK 121 NWFVALKKNG SCKLGPRTHYGQKAILFLPL PVSSD Amino acid sequence of Meleagris gallopavo (turkey)FGF1(SEQ ID NO: 58) (Ensembl accession no. ENSMGAP00000016398; partialsequence corresponding to human FGF1 residues 1 to 56, which is herebyincorporated by reference in its entirety): 1 MAEGEITTFT ALTERFGLPLGNYKKPKLLY CSNGGHFLRI LPDGKVDGTR DRSDQH Amino acid sequence of Macropuseugenii (wallaby) FGF1(SEQ ID NO: 59) (Ensembl accession no.ENSMEUP00000015084, which is hereby incorporated by reference in itsentirety): 1 MAEGEITTFT ALTERFNLPL GNYKKPKLLY CSNGGHFLRI LPDGKVDGTRDRNDQHIQLQ 61 LSAESVGEVY IKSTESGQYL AMDTNGLLYG SQTPSEECLF LERLEENHYNTYISKKHAEK 121 NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSE Amino acidsequence of Danio rerio (zebrafish) FGF1(SEQ ID NO: 60) (Ensemblaccession no. ENSDARP00000008825, which is hereby incorporated byreference in its entirety): 1 MTEADIAVKS SPRDYKKLTR LYCMNGGFHLQILADGTVAG AADENTYSIL RIKATSPGVV 61 VIEGSETGLY LSMNEHGKLY ASSLVTDESYFLEKMEENHY NTYQSQKHGE NWYVGIKKNG 121 KMKRGPRTHI GQKAIFFLPR QVEQEED

As noted above, the portion of the paracrine FGF may be modified todecrease binding affinity for heparin and/or heparan sulfate compared tothe portion without the modification. In one embodiment, the modifiedportion of the paracrine FGF includes one or more substitutions,additions, or deletions.

In one embodiment, the one or more substitutions are located at one ormore amino acid residues of SEQ ID NO: 1 selected from N33, K127, K128,N129, K133, R134, R137, Q142, K143, and combinations thereof. In oneembodiment, the one or more substitutions are selected from N33T, K127D,K128Q, N129T, K133V, R134L, R137H, Q142M, K143T/L/I, and combinationsthereof. In one embodiment, the modification is one or moresubstitutions which are located at one or more amino acid residuescorresponding to residues of SEQ ID NO: 1 selected from N33, K127, K128,N129, K133, R134, R137, Q142, K143, and combinations thereof. In oneembodiment, the modification is one or more substitutions which arelocated at one or more amino acid residues corresponding to residues ofSEQ ID NO: 1 selected from N33, K127, K128, N129, K133, R134, R137,Q142, K143, and combinations thereof. Amino acid residues correspondingto those of SEQ ID NO: 1 may be determined by, for example, sequenceanalysis and structural analysis.

Also encompassed within the present invention are portions of paracrineFGFs other than FGF1 (e.g., FGF2, FGF4, FGF5, FGF6, FGF9, FGF16, andFGF20). The portions derived from paracrine FGFs other than FGF1 includeportions corresponding to the above-identified amino acid sequences ofFGF1. Corresponding portions may be determined by, for example, sequenceanalysis and structural analysis.

It will be understood that the portion of the paracrine FGF according tothe present invention may be derived from a nucleotide sequence thatencodes a paracrine FGF protein. For example, in one embodiment, thenucleotide sequence is the nucleotide sequence that encodes human FGF1(GenBank Accession No. BC032697, which is hereby incorporated byreference in its entirety) (SEQ ID NO: 61), as follows:

91                                  ATGGCTGAAG GGGAAATCAC CACCTTCACA 121GCCCTGACCG AGAAGTTTAA TCTGCCTCCA GGGAATTACA AGAAGCCCAA ACTCCTCTAC 181TGTAGCAACG GGGGCCACTT CCTGAGGATC CTTCCGGATG GCACAGTGGA TGGGACAAGG 241GACAGGAGCG ACCAGCACAT TCAGCTGCAG CTCAGTGCGG AAAGCGTGGG GGAGGTGTAT 301ATAAAGAGTA CCGAGACTGG CCAGTACTTG GCCATGGACA CCGACGGGCT TTTATACGGC 361TCACAGACAC CAAATGAGGA ATGTTTGTTC CTGGAAAGGC TGGAGGAGAA CCATTACAAC 421ACCTATATAT CCAAGAAGCA TGCAGAGAAG AATTGGTTTG TTGGCCTCAA GAAGAATGGG 481AGCTGCAAAC GCGGTCCTCG GACTCACTAT GGCCAGAAAG CAATCTTGTT TCTCCCCCTG 541CCAGTCTCTT CTGATTAA

In another embodiment of the present invention, the portion of theparacrine FGF of the chimeric protein may be derived from a nucleotidesequence that encodes an ortholog of human FGF1. Nucleotide sequencesthat encode FGF1 orthologs are shown in Table 2.

TABLE 2 Olive Baboon FGF1 gene coding sequence (1-155) (SEQ ID NO: 62)(GenBank accession no. NM_001169086, which is hereby incorporated byreference in its entirety): 1 ATGGCTGAAG GGGAAATCAC CACGTTCACAGCCCTGACCG AGAAGTTTAA TCTGCCTCCA 61 GCGAATTACA AGAAGCCCAA ACTGCTCTACTGTAGCAACG GGGGACACTT CTTGAGGATC 121 CTTCCGGATG GCACAGTGGA TGGGACAAGGGACAGGAGCG ACCAGCACAT TCAGCTGCAG 181 CTCAGTGCGG AAAGCGTGGG GGAGGTGTATATAAAGAGTA CCGAGACTGG CCAGTACTTG 241 GCCATGGACA CCGACGGGCT TTTATACGGCTCACAGACAC CAAATGAGGA ATGTTTGTTC 301 CTGGAAAGGC TGGAGGAGAA CCATTACAACACCTACATAT CCAAGAAGCA CGCAGAGAAG 361 AATTGGTTTG TTGGCCTCAA GAAGAATGGAAGCTGCAAAC GTGGTCCTCG GACTCACTAT 421 GGCCAGAAAG CAATCTTGTT TCTTCCCCTGCCAGTCTCTT CTGATTAA Sumatran orangutan FGF1 gene coding sequence(60-214) (SEQ ID NO: 63) (GenBank accession no. NM_001133601, which ishereby incorporated by reference in its entirety): 211                                 ATGGCTGAAG GGGAAATCAC CACCTTCACA 241GCCCTGACCG AGAAGTTTAA TCTGCCTCCA GGGAATTACA AGAAGCCCAA ACTCCTCTAC 301TGTAGCAACG GGGGCCACTT CTTGAGGATC CTTCCGGATG GCACAGTGGA TGGGACAAGG 361GACAGGAGCG ACCAGCACAT TCAGCTGCAG CTCAGTGCGG AAAGCGTGGG GGAGGTGTAT 421ATAAAGAGTA CCGAGACTGG CCAGTACTTG GCCATGGACA CCGACGGGCT TTTATACGGC 481TCACAGACAC CAAATGAGGA ATGTTTGTTC CTGGAAAGGC TGGAGGAGAA CCATTACAAC 541ACCTATATAT CCAAGAAGCA TGCAGAGAAG AATTGGTTTG TTGGCCTCAA GAAGAATGGA 601AGCTGCAAAC GCGGTCCTCG GACTCACTAT GGCCAGAAAG CAATCTTGTT TCTCCCCCTG 661CCAGTCTCTT CCGATTAA White-tufted-ear marmoset FGF1 gene coding sequence(1-155) (SEQ ID NO: 64) (GenBank accession no. XM_002744295, which ishereby incorporated by reference in its entirety): 130          ATGGCTGAAGG GGAAATCACC ACCTTCACAG CCCTGACCGA GAAGTTTGAT 181 CTGCCTCCAGGGAATTACAA GAAGCCCAAA CTCCTCTACT GTAGCAATGG GGGCCACTTC 241 TTGAGGATCCTTCCGGATGG CACAGTGGAT GGGACAAGGG ACAGGAGCGA CCAGCACATT 301 CAGCTGCAGCTCAGTGCGGA AAGCGTGGGG GAGGTGTATA TAAAGAGTAC CGAGACTGGC 361 CAGTACTTGGCCATGGACAC CGACGGGCTT TTATACGGCT CACAGACACC AAATGAGGAA 421 TGTTTGTTCCTGGAGAGGCT GGAGGAGAAC CATTACAACA CCTATATATC CAAGAAACAT 481 GCAGAGAAGAATTGGTTTGT CGGCCTCAAG AAGAATGGAA GCTGTAAACG TGGTCCTCGG 541 ACTCACTATGGTCAGAAAGC GATCTTGTTT CTCCCCCTGC CAGTTTCTTC TGATTAA Horse FGF1 genecoding sequence (1-155) (SEQ ID NO: 65) (GenBank accession no.NM_001163886, which is hereby incorporated by reference in itsentirety): 34                                     ATGGCTG AAGGAGAAATCACAACCTTC 61 ACGGCCCTGA CCGAGAAGTT TAATCTGCCT CCAGGGAATT ACAAGAAGCCCAAACTCCTC 121 TACTGTAGCA ATGGGGGCCA CTTCCTGAGG ATCCTTCCAG ATGGCACAGTGGATGGGACA 181 AGGGACAGGA GCGACCAGCA CATTCAGCTG CAGCTCAGTG CGGAAAGCGTGGGGGAGGTG 241 TATATAAAGA GTACCGAGAC TGGCCAGTAC TTGGCCATGG ACACCGACGGGCTGTTGTAC 301 GGCTCACAGA CACCAAACGA GGAATGTTTG TTCCTGGAAA GGCTGGAGGAAAACCATTAC 361 AACACCTACA CATCCAAGAA GCATGCAGAG AAGAACTGGT TCGTTGGTCTCAAGAAGAAT 421 GGGAGCTGCA AACGCGGTCC TCGGACTCAC TATGGGCAGA AAGCAATCTTGTTTCTTCCC 481 CTGCCCGTCT CCTCTGACTA A Chimpanzee FGF1 gene codingsequence (1-155) (SEQ ID NO: 66) (GenBank accession no. GABD01003589,which is hereby incorporated by reference in its entirety): 80                    A TGGCTGAAGG GGAAATCACC ACCTTCACAG CCCTGACCGA 121GAAGTTTAAT CTGCCTTCAG GGAATTACAA GAAGCCCAAA CTCCTCTACT GTAGCAACGG 181GGGCCACTTC CTGAGGATCC TTCCGGATGG CACAGTGGAT GGGACAAGGG ACAGGAGCGA 241CCAGCACATT CAGCTGCAGC TCAGTGCGGA AAGCGTGGGG GAGGTGTATA TAAAGAGTAC 301CGAGACTGGC CAGTACTTGG CCATGGACAC CGACGGGCTT TTATACGGCT CACAGACACC 361AAATGAGGAA TGTTTGTTCC TGGAACGGCT GGAGGAGAAC CATTACAACA CCTATATATC 421CAAGAAGCAT GCAGAGAAGA ATTGGTTTGT TGGCCTCAAG AAGAATGGAA GCTGCAAACG 481CGGTCCTCGG ACTCACTATG GCCAGAAAGC AATCTTGTTT CTCCCCCTGC CAGTCTCTTC 541CGATTAA Elephant FGF1 gene coding sequence (1-155) (SEQ ID NO: 67)(GenBank accession no. XM_003404573, which is hereby incorporated byreference in its entirety): 1 ATGGCCGAAG GGGAAATCAC AACTTTCACAGCCCTGACAG AGAAGTTCAA CCTGCCTCCA 61 GGGAATTACA AGAAGCCCAA ACTCCTCTACTGTAGCAATG GAGGTCACTT CTTAAGGATC 121 CTTCCAGATG GCACAGTGGA TGGCACCAGGGACAGGAGTG ACCAGCACAT TCAGCTGCAG 181 CTCAGTGCGG AAAGCGTGGG GGAGGTGTATATAAAGGGCA CCGAGACTGG CCAGTACTTG 241 GCCATGGACA CCGACGGGCT TTTATACGGCTCACAGACAC CAAATGAGGA ATGTTTGTTC 301 CTGGAAAGGC TGGAGGAAAA CCATTACAACACCTACACAT CCAAGAAGCA CGCAGAGAAG 361 AATTGGTTCG TTGGTCTCAA GAAGAATGGAAGCTGCAAAC GCGGTCCTCG GACTCACTAT 421 GGCCAGAAAG CAATCTTGTT TCTCCCCCTGCCAGTCTCCT CTGATTAA Dog FGF1 gene coding sequence (1-155) (SEQ ID NO:68) (GenBank accession no. XM_844181, which is hereby incorporated byreference in its entirety): 164                                               ATGGCTG AAGGGGAAAT 181CACAACCTTC ACTGCCCTGA CGGAGAAGTT TAATCTGCCT CCGGGGAATT ACATGAAGCC 241CAAACTCCTC TACTGTAGCA ACGGGGGCCA CTTCCTGAGG ATCCTTCCAG ATGGCACAGT 301GGATGGGACA AGGGACAGGA GCGACCAGCA CATTCAGCTG CAGCTCAGCG CGGAAAGCGT 361GGGGGAGGTG TATATAAAGA GCACCGAGAC TGGCCAGTAC TTGGCCATGG ACACCGATGG 421GCTTCTGTAC GGCTCACAGA CACCGAATGA GGAATGTTTG TTCCTGGAAA GGCTGGAGGA 481AAACCATTAC AACACCTACA CATCCAAGAA GCATGCAGAA AAAAATTGGT TTGTTGGTCT 541CAAGAAGAAT GGAAGCTGCA AACGCGGTCC TCGGACTCAC TATGGTCAAA AAGCAATTTT 601GTTTCTCCCC CTGCCAGTGT CCTCTGATTA A Giant panda FGF1 gene coding sequence(1-155) (SEQ ID NO: 69) (GenBank accession no. XM_002912535, which ishereby incorporated by reference in its entirety): 146                           ATGGC TGAAGGGGAG ATCACAACCT TCACCGCCCT 181GACGGAGAAG TTTAATCTGC CTGCGGGGAA TTACAAGAAG CCCAAACTCC TCTACTGTAG 241CAACGGGGGC CACTTCCTGA GGATCCTTCC AGATGGCACA GTGGACGGGA CGAGGGACAG 301GAGCGACCAG CACATTCAAC TGCAGCTCAG CGCGGAAAGC GTAGGGGAGG TGTACATAAA 361GAGCACCGAG ACCGGCCAGT ACTTGGCCAT GGACACCGAT GGGCTTCTGT ACGGCTCACA 421GACACCAAAT GAGGAATGTT TGTTCCTGGA AAGGCTGGAG GAAAACCATT ACAACACCTA 481CACATCCAAG AAGCACGCGG AGAAGAATTG GTTTGTTGGT CTCAAGAAGA ATGGAAGCTG 541CAAACGTGGT CCTCGGACTC ACTATGGCCA GAAAGCAATT CTGTTTCTCC CCCTGCCAGT 601CTCCTCTGAT TAA Bolivian squirrel monkey FGF1 gene coding sequence(1-155) (SEQ ID NO: 70) (GenBank accession no. XM_003920547, which ishereby incorporated by reference in its entirety): 130          ATGGCTGAAGG GGAAATCACC ACCTTTACAG CCCTGACCGA GAAGTTTGAT 181 CTGCCTCCAGGGAATTACAA GAAGCCCAAA CTCCTCTACT GTAGCAACGG GGGCCACTTC 241 TTGAGGATCCTTCCGGATGG CACAGTGGAT GGGACCAGGG ACAGGAGCGA TCTTCACATT 301 CAGCTGCAGCTCAGTGCGGA AAGCGTGGGG GAGGTGTATA TAAAGAGTAC CGAGACTGGC 361 CAGTACTTGGCCATGGACAC CGACGGGCTT TTATACGGCT CACAGACACC AAATGAGGAA 421 TGTTTGTTCCTGGAAAGGCT GGAGGAGAAC CATTACAACA CCTATATATC CAAGAAACAC 481 GCAGAGAAGAATTGGTTTGT TGGCCTCAAG AAGAATGGAA GCTGCAAGCG CGGTCCTCGG 541 ACTCACTATGGCCAGAAAGC AATCTTGTTT CTCCCCCTGC CAGTCTCTTC TGATTAA Pig FGF1 gene codingsequence (1-155) (SEQ ID NO: 71) (GenBank accession no. XM_003124010,which is hereby incorporated by reference in its entirety): 35                                     ATGGCT GAAGGCGAAA TCACAACCTT 61CACGGCCCTG ACCGAGAAGT TTAATCTGCC TCCAGGAAAT TACAAGAAGC CCAAGCTCCT 121CTACTGCAGC AACGGGGGCC ATTTCCTCAG GATCCTTCCA GATGGCACAG TGGATGGGAC 181CAGGGACAGG AGCGACCAGC ACATTCAGCT GCAGCTCAGT GCGGAAAGCG TGGGGGAGGT 241GTATATAAAG AGTACGGAGA CTGGCCAGTA CTTGGCCATG GACACCAGCG GGCTTTTGTA 301CGGCTCACAG ACACCCAGTG AGGAGTGTTT GTTCCTGGAG AGGCTGGAGG AAAACCATTA 361CAATACCTAC ACATCCAAGA AGCACGCAGA GAAGAACTGG TTCGTTGGCC TCAAGAAGAA 421TGGAAGCTGC AAACGCGGTC CTCGGACTCA CTATGGCCAG AAAGCCATCC TGTTTCTCCC 481CCTGCCAGTA TCCTCGGATT AA Small-eared galago FGF1 gene coding sequence(1-155) (SEQ ID NO: 72) (GenBank accession no. XM_003782087, which ishereby incorporated by reference in its entirety): 28                             ATG GCTGAAGGGG AAATCACAAC CTTCACAGCC 61CTCACAGAGA AGTTTAATCT GCCTCTAGGA AATTACAAGA AGCCCAAGCT CCTCTACTGT 121AGCAACGGGG GTCACTTTCT GAGGATCCTG CCGGATGGCA CCGTGGATGG GACACAAGAC 181AGGAGCGACC AGCACATTCA GCTGCAGCTC AGTGCGGAAA GCGTGGGGGA GGTGTATATA 241AAGAGTACCC AGACTGGCCA GTACTTGGCC ATGGACTCCG ACGGGCTTTT ATACGGCTCA 301CAAACACCAA ATGAGGAATG CCTGTTCCTG GAACGGCTGG AGGAAAACCA TTACAACACC 361TATGTGTCCA AGAAGCACGC CGAGAAGAAT TGGTTTGTCG GTCTCAAGAA GAACGGAAGT 421TGCAAACGTG GTCCTCGGAC TCACTACGGC CAGAAAGCAA TCTTGTTTCT CCCCCTGCCA 481GTCTCCTCTG ATTAA Greater horseshoe bat FGF1 gene coding sequence (1-155)(SEQ ID NO: 73) (GenBank accession no. DP000705, which is herebyincorporated by reference in its entirety): 190120                                          T TAATCAGAGG AGACTGGCAG 190141GGGGAGAAAC AGGATTGCTT TCTGGCCATA GTGAGTCCGA GGACCGCGCT TGCAGCTTCC 190201ATTCTTCTTG AGCCCAACGA ACCAATTCTT TTCTGCGTGC TTCTTGGACG TGTAGGTGTT 190261GTAATGGTTT TCCTCCAGCC TTTCCAGGAA CAGACATTCC TCATTTGGTG TCTG 194466     TGAGC CGTACAAAAG CCCGTCGGAG TCCATGGCCA AGTACTGGCC ACTCTCGGTG 194521CTCTTTATAT ACACCTCCCC CACGCTTTCC GCACTGAGCT GCAGCTGAA 208114                                    TGTGCTG GTCACTCTTG TCCCTTGTCC 208141CATCCACTGT GCCATCTGGA AGGATCCTCA GGAAGTGGCC CCCGTTGCTG CAGTAGAGAA 208201GTTTGGGTTT CTTGTAATTC CCTGTAGGCA GATTAAACTT CTCAGTAAGG GCTGTGAACG 208261TGGTGACTTC CCCTTCGGCC AT European shrew FGF1 gene coding sequence(1-155) (SEQ ID NO: 74) (GenBank accession no. DP000767, which is herebyincorporated by reference in its entirety): 138344                                               CTAGTCG GAGGAGACGG 138361GCAGGGGGAG AAACAAGATC GCTTTCTGGC CGTAGTGAGT CCGGGGACCA CGCTTGCAGC 138421TTCCGTTCTT CTTCAGACCA ACAAACCAAT TCTTCTCGGC ATGCTTCTTG GAGGTATAGG 138481TGTTGTAATG GTTTTCCTCC AGCCTTTCCA GAAACAGACA TTCCTCATTC GGTGTTTG 143512                                                        TGAGCCGTA 143521TAAAAGCCCG TCGGTGTCCA TGGCCAAGTA ATGGCCAGTC TCCGTGCTCT TTATATACAC 143581CTCCCCCACG CTTTCCGCAC TGAGCTGCAG CTGAA 157009                                                    TG TGCTGGTCGC 157021TGCGGTCCCT GGTCCCATCC ACTGTGCCGT CCGGGAGGAT GCGCAGGAAG TGGCCCCCGT 157081TGCTGCAGTA CAGGAGTTTG GGCTTCTTGT AGTTCCCTGG TGGCAGGTTA AACTTCTCCA 157141TGAGGGCCCC AAAGGTGGTG ATCTCCCCCT CGGCCAT Rabbit FGF1 gene codingsequence (1-155) (SEQ ID NO: 75) (GenBank accession no. NM_001171488,which is hereby incorporated by reference in its entirety): 1 ATGGCTGAGGGGGAGGTCAC CACCTTCACA GCCCTGACCG AGAAGTTCAA CCTGCCTGCA 61 GGGAACTACAAGTTGCCCAA ACTCCTCTAC TGCAGCAACG GGGGCCACTT CCTGAGGATC 121 CTGCCGGACGGCACTGTGGA CGGCACAAGG GACAGGAGCG ACCAGCACAT TCAGCTGCAG 181 CTGAGTGCGGAAAGCGTGGG GGAGGTGTAT ATAAAGAGTA CGGAGACCGG CCAGTACTTG 241 GCCATGGACACCGACGGCCT TTTATACGGC TCGCAAACGC CCAGTGAGGA GTGTTTGTTC 301 CTGGAACGGCTGGAGGAGAA CCACTACAAC ACCTACACGT CCAAGAAGCA CGCCGAGAAG 361 AACTGGTTCGTGGGGCTGAA GAAAAACGGG AGCTGCAAGC GCGGTCCTCG GACTCACTAC 421 GGCCAGAAAGCCATCTTGTT CCTCCCCCTG CCGGTCTCCT CCGACTAA Chinese hamster FGF1 genecoding sequence (1-155) (SEQ ID NO: 76) (GenBank accession no.XM_003502421, which is hereby incorporated by reference in itsentirety): 1 ATGGCTGAAG GAGAAATCAC CACCTTCTCA GCCCTGACAG AGAGATTTAATCTGCCTCCA 61 GGAAACTACA AGAAGCCCAA ACTGCTCTAC TGCAGCAACG GGGGCCACTTCTTGAGGATC 121 CTTCCAGATG GCACAGTGGA TGGGACAAGG GACAGGAGTG ACCAGCACATTCAGCTGCAG 181 CTGAGTGCGG AAAGCGCGGG CGAAGTGTAT ATAAAGGGTA CAGAGACAGGCCAGTACAGG 241 AACATGGACA CGGATGGCCT TTTATACGGC TCACAGACAC CAAATGAAGAATGCCTGTTC 301 CTGGAAAGGC TGGAAGAAAA CCATTACAAC ACTTATACAT CCAAGAAGCACGCAGAGAAG 361 AACTGGTTTG TGGGCCTCAA GAAAAACGGG AGCTGCAAGC GTGGTCCTCGGACTCACTAT 421 GGCCAGAAAG CAATCTTGTT TCTCCCCCTG CCTGTATCTT CTGACTAGTasmanian devil FGF1 gene coding sequence (1-155) (SEQ ID NO: 77)(GenBank accession no. XM_003756690, which is hereby incorporated byreference in its entirety): 24                          ATGGCCGAAGGGGAGAT CACAACCTTC ACAGCCCTGA 61 CCGAAAGATT TAATCTGCCA CTGGGGAATTACAAGAAGCC CAAGCTTCTC TACTGTAGCA 121 ATGGGGGCCA CTTTTTGAGG ATTCTTCCTGATGGTAAAGT GGATGGGACA AGGGACAGAA 181 ATGATCAACA CATTCAACTG CAACTAAGCGCGGAAAGCGT GGGTGAGGTG TATATAAAGA 241 GCACTGAGTC TGGCCAGTAT TTGGCTATGGACACCGATGG ACTTTTATAC GGCTCACAGA 301 CACCCACTGA AGAATGCTTG TTCCTGGAGAGATTGGAGGA GAATCATTAC AACACCTACA 361 TATCAAAGAA GCATGCGGAG AAAAATTGGTTTGTGGGCCT CAAGAAAAAT GGAAGCTGCA 421 AAAGAGGTCC CAGGACTCAC TATGGCCAGAAAGCCATCCT CTTCCTTCCC CTCCCTGTGT 481 CCTCTGAGTA A House mouse FGF1 genecoding sequence (1-155) (SEQ ID NO: 78) (GenBank accession no.NM_010197, which is hereby incorporated by reference in its entirety):188        ATG GCTGAAGGGG AGATCACAAC CTTCGCAGCC CTGACCGAGA GGTTCAACCT241 GCCTCTAGGA AACTACAAAA AGCCCAAACT GCTCTACTGC AGCAACGGGG GCCACTTCTT301 GAGGATCCTT CCTGATGGCA CCGTGGATGG GACAAGGGAC AGGAGCGACC AGCACATTCA361 GCTGCAGCTC AGTGCGGAAA GTGCGGGCGA AGTGTATATA AAGGGTACGG AGACCGGCCA421 GTACTTGGCC ATGGACACCG AAGGGCTTTT ATACGGCTCG CAGACACCAA ATGAGGAATG481 TCTGTTCCTG GAAAGGCTGG AAGAAAACCA TTATAACACT TACACCTCCA AGAAGCATGC541 GGAGAAGAAC TGGTTTGTGG GCCTCAAGAA GAACGGGAGC TGTAAGCGCG GTCCTCGGAC601 TCACTATGGC CAGAAAGCCA TCTTGTTTCT GCCCCTCCCG GTGTCTTCTG ACTAGDomestic guinea pig FGF1 gene coding sequence (1-154) (SEQ ID NO: 79)(GenBank accession no. XM_003477194, which is hereby incorporated byreference in its entirety): 1 ATGGCTGAAG GAGAAATCAC AACTTTTGCAGCCCTGACTG AGAAGTTTAA TCTGCCTCCA 61 GGGAATTATA AGAAGCCCAA ACTGCTCTACTGCAGCAATG GGGGCCACTT CCTGAGGATC 121 CTTCCAGACG GCACAGTGGA CGGCACAAGAGACAGGAGCG ACCAGCACAT TCAGCTGCAG 181 CTCAGTGCGG AAGGCGTGGG GGAGGTGTATATACAGAGCA CCGAGACCGG CCAGTACTTG 241 GCCATGGACA CCGACGGGCT TTTATACGGCTCACAGACAC CAAGTGAGGA ATGCTTGTTC 301 CTGGAAAGGC TGGAGGAAAA CCATTACAACACCTACACAT CCAAGAAGCA TGTGGAGAAG 361 AATTGGTTTG TTGGCCTCAA GAAGAACGGAAGCTGCAAGC GTGGTCCTCG GACTCACTAT 421 GGCCAGAAAG CAATCTTGTT CCTCCCCTTGCCAGTCTCTG ATTAG Gray short-tailed opossum FGF1 gene coding sequence(1-155) (SEQ ID NO: 80) (GenBank accession no. XM_001368884, which ishereby incorporated by reference in its entirety): 1 ATGGCCGAAGGGGAGATCAC AACCTTCACA GCCCTGACTG AAAGATTTAA CCTGCCACTG 61 GGGAATTACAAGAAACCCAA GCTTCTCTAC TGTAGCAATG GGGGCCATTT CTTGAGGATC 121 CTTCCTGATGGCAAAGTGGA TGGGACACGG GACAGAAATG ATCAACACAT TCAACTGCAG 181 CTGAGCACGGAAAGTGTGGG TGAGGTGTAT ATAAAGAGCA CTGAGTCTGG CCAGTATTTG 241 GCTATGGACACCGATGGACT TTTATATGGC TCACAGACAC CCAGTGAAGA ATGCTTGTTT 301 CTGGAGAGGTTGGAGGAGAA TCATTACAAC ACCTACACAT CGAAGAAGCA TGCAGAGAAA 361 AATTGGTTTGTTGGTCTCAA GAAGAATGGA AGCTGCAAAA AGGGTCCCAG GACTCACTAC 421 GGCCAGAAAGCCATCCTGTT CCTTCCCCTC CCTGTGTCCT CTGAGTAA Common vampire bat FGF1 genecoding sequence (1-155) (SEQ ID NO: 81) (GenBank accession no.GABZ01008334, which is hereby incorporated by reference in itsentirety): 1 ATGGCTGAAG GGGAAGTCAC CACGTTCACA GCTCTGACTG AGAAGTTTAATCTGCCTCTG 61 GAGAGTTACA AGAAGCCCAA ACTTCTCTAC TGCAGCAACG GTGGCCACTTCCTGAGGATC 121 CTTCCAGATG GTACAGTGGA TGGGACAAGG GACAAGAGCG ACCAGCACATTCAGCTGCAG 181 CTCAGTGCGG AAAGCGTGGG GGAGGTGTAC ATAAAGAGCA CCGGGAGTGGCCAGTACTTG 241 GCCATGGACT CCGCCGGGCT TTTGTATGGC TCACAGACAC CAAATGAGGAATGTTTGTTC 301 CTGGAAAGGC TGGAGGAAAA CCATTACAAC ACCTACACAT CCAAGAAGCATGCAGAAAAG 361 AATTGGTTCG TGGGGCTCAA GAAGAATGGA AGCTGCAAGC GTGGCCCCCGGACTCATTAT 421 GGCCAGAAAG CAATCTTGTT TCTCCCCCTG CCAGTCAACT CTGATTAACattle FGF1 gene coding sequence (1-155) (SEQ ID NO: 82) (GenBankaccession no. NM_174055, which is hereby incorporated by reference inits entirety): 918                   ATG GCTGAAGGAG AAACCACGACCTTCACGGCC CTGACTGAGA 961 AGTTTAACCT GCCTCTAGGC AATTACAAGA AGCCCAAGCTCCTCTACTGC AGCAACGGGG 1021 GCTACTTCCT GAGAATCCTC CCAGATGGCA CAGTGGATGGGACGAAGGAC AGGAGCGACC 1081 AGCACATTCA GCTGCAGCTC TGTGCGGAAA GCATAGGGGAGGTGTATATT AAGAGTACGG 1141 AGACTGGCCA GTTCTTGGCC ATGGACACCG ACGGGCTTTTGTACGGCTCA CAGACACCCA 1201 ATGAGGAATG TTTGTTCCTG GAAAGGTTGG AGGAAAACCATTACAACACC TACATATCCA 1261 AGAAGCATGC AGAGAAGCAT TGGTTCGTTG GTCTCAAGAAGAACGGAAGG TCTAAACTCG 1321 GTCCTCGGAC TCACTTCGGC CAGAAAGCCA TCTTGTTTCTCCCCCTGCCA GTCTCCTCTG 1381 ATTAA Platypus FGF1 gene coding sequence(1-155) (SEQ ID NO: 83) (GenBank accession no. XM_001514811, which ishereby incorporated by reference in its entirety): 1 ATGGCGGAGGGTGAAATCAC CACGTTCACA GCCCTGATGG AGAAGTTCGA CCTACCCCTG 61 GGCAACTACAAAAAGCCTAG GCTGCTCTAC TGCAGCAATG GCGGCTACTT CCTGCGCATC 121 CAGCCAGACGGTAAAGTGGA CGGGACCAGG GATCGGAGCG ATCAGCACAT TCAACTGCAG 181 CTAAGCGCGGAAAGCGTGGG CGAGGTGTAT ATAAAGAGCA CCGAGTCTGG CCACTATTTG 241 GCTATGGACACCGAAGGACT TTTATATGGC TCACAGGCAC CCAGTGAAGA CTGCTTGTTC 301 CTGGAGCGGCTGGAGGAGAA CCACTATAAC ACGTACGTGT CCAAGAAGCA CGCTGAGAAG 361 AATTGGTTTGTCGGTCTCAA GAAGAACGGG AGCTGCAAAC GAGGTCCCCG GACTCACTAC 421 GGCCAGAAAGCCATCCTCTT CCTCCCGCTC CCCGTGGCAT CCGACTAG Zebra finch FGF1 gene codingsequence (1-155) (SEQ ID NO: 84) (GenBank accession no. XM_002193251,which is hereby incorporated by reference in its entirety): 1 ATGGCCGAGGGGGAGATCAC CACCTTCAGC GCCCTGACGG AGAAGTTCAA CCTGCCCCCG 61 GGGAACTACAAGAAGCCCAA ACTGCTGTAC TGCAGCAACG GGGGGCATTT CCTGCGCATC 121 CTCCCGGACGGCACCGTGGA TGGCACCAGG GACCGCAGCG ACCAGCACAT TCAGCTCCAG 181 CTGAGTGCAGAGAGCGTGGG GGTGGTGCAC ATCCAGAGCA CCCAGTCGGG GCAGTACCTG 241 GCCATGGACACCAACGGGCT GCTCTACGGC TCGCAGCTGC CACCCGGTGA GTGTCTGTTC 301 CTGGAAAGGCTGGAGGAGAA CCATTACAAC ACCTACGTCT CCAAAATGCA CGCGGACAAG 361 AACTGGTTTGTGGGGCTGAA GAAGAACGGG ACAAGCAAGC TGGGCCCGCG GACTCACTAC 421 GGCCAGAAGGCGATCCTGTT CCTGCCGCTG CCCGTGGCGG CCGACTGA Nine-banded armadillo FGF1gene coding sequence (1-155) (SEQ ID NO: 85) (GenBank accession no.DP001080, which is hereby incorporated by reference in its entirety):178389         TT AATCAGAGGA GACTGGCAGG GGAAGAAACA AGATAGCTTT CTGGCCATAG178441 TGAGTCTGAG GACCACGTTT GCTGCTTCCG TCCTTCTTGA GACCAACAAA CCATTTCTTC178501 TCTGCATGCT TCTTGGATAT GTAGGTGTTG TAATTGTTTT CTTCCAGCTT TTCCATGAAC178561 AAGCATTCCT CACTTGGTGT CTC 182873                                                         TGAGCCAT 182881ATAAAAGCCC GTCGGTGTCC ATGGCTAAGT ACTGGCCGGT CTCTGCACTC TTTATATACA 182941CCTCCCCCAC GCTTTCCGCA CTGAGCTGCA GCTGAA 197786                           TGTGT TGGTCGCTCC TGTCCCTTGT CCCATCCACC 197821GTGCCATCTG GAAGGATCCT CAAGAAGTGG CCCCCGTTTC TGCAGTAGAG GAGTCTGGGG 197881TGCTTGTAAT TTTCTAGGGG CAGGTTGAAC TTCTCCATCA GGGCCATGAA GGTTGTGATC 197941TCCCCTTCAG CCAT Xenopus Silurana tropicalis FGF1 gene coding sequence(1-155) (SEQ ID NO: 86) (GenBank accession no. FJ428265, which is herebyincorporated by reference in its entirety): 1 ATGGCAGAGG GAGACATCACAACATTCAAC CCCATTGCAG AGTCCTTCAG TCTTCCAATT 61 GGCAACTACA AGAAACCAAAACTTCTGTAC TGTAATAATG GAGGGTATTT TTTGCGCATC 121 CTCCCAGATG GGGTTGTGGATGGAACAAGA GACAGAGATG ACCTTTACAT TACACTGAAG 181 TTAAGCGCAC AAAGCCAAGGGGAGGTGCAT ATCAAAAGCA CAGAGACAGG GAGTTACTTA 241 GCCATGGACT CCAGTGGACAGTTGTATGGA ACTCTCACAC CAAATGAAGA AAGCCTGTTT 301 CTGGAGACAT TAGAAGAGAATCACTATAAC ACATACAAGT CAAAGAAGTA TGCAGAAAAT 361 AACTGGTTTG TGGGGATAAAGAAGAACGGG GCAAGCAAAA AGGGATCAAG GACTCACTAT 421 GGACAAAAAG CCATCCTTTTTCTGCCGCTG CCAGCATCAC CTGACTAG Heterocephalus glaber FGF1 gene codingsequence (1-155) (SEQ ID NO: 87) (generated using SMS Reverse Translatetool on the ExPASy Bioinformatics Resource website (www.expasy.org): 1ATGGCGGAAG GCGAAATTAC CACCTTTACC GCGCTGACCG AAAAATTTAA CCTGCCGCCG 61GGCAACTATA AAAAACCGAA ACTGCTGTAT TGCAGCAACG GCGGCCATTT TCTGCGCATT 121CTGCCGGATG GCAAAGTGGA TGGCACCCGC GATCGCAGCG ATCAGCATAT TCAGCTGCAG 181CTGAGCGCGG AAGGCGTGGG CGAAGTGTAT ATTAAAAGCA CCGAAACCGG CCAGTATCTG 241GCGATGGATA CCGATGGCCT GCTGTATGGC AGCCAGACCG CGAGCGAAGA ATGCCTGTTT 301CTGGAACGCC TGGAAGAAAA CCATTATAAC ACCTATATTA GCAAAAAACA TGCGGAAAAA 361AACTGGTTTG TGGGCCTGAA AAAAAACGGC AGCTGCAAAC GCGGCCCGCG CACCCATTAT 421GGCCAGAAAG CGATTCTGTT TCTGCCGCTG CCGGTGAGCA GCGAT Black flying fox FGF1gene coding sequence (1-155) (SEQ ID NO: 88) (generated using SMSReverse Translate tool on the ExPASy Bioinformatics Resource website(www.expasy.org): 1 ATGGCGGAAG GCGAAGTGAC CACCTTTACC GCGCTGACCGAACGCTTTAA CCTGCCGCCG 61 GGCAACTATA AAAAACCGAA ACTGCTGTAT TGCAGCAACGGCGGCCATTT TCTGCGCATT 121 CTGCCGGATG GCACCGTGGA TGGCACCCGC GATAAAAGCGATCAGCATAT TCAGCTGCAG 181 CTGAGCGCGG AAAGCGTGGG CGAAGTGTAT ATTAAAAGCACCGAAAGCGG CCAGTATCTG 241 GCGATGGATA GCGATGGCCT GCTGTATGGC AGCCAGACCCCGGATGAAGA TTGCCTGTTT 301 CTGGAACGCC TGGAAGAAAA CCATTATAAC ACCTATACCAGCAAAAAACA TGCGGAAAAA 361 AACTGGTTTG TGGGCCTGAA AAAAAACGGC AGCTGCAAACGCGGCCCGCG CACCCATTAT 421 GGCCAGAAAG CGATTCTGTT TCTGCCGCTG CCGGTGAGCAGCGAT Chinese tree shrew FGF1 gene coding sequence (1-155) (SEQ ID NO:89) (generated using SMS Reverse Translate tool on the ExPASyBioinformatics Resource website (www.expasy.org): 1 ATGGCGGAAGGCGAAATTAC CACCTTTGCG GCGCTGACCG AAAAATTTGA TCTGCCGCCG 61 GGCAACTATAAAAAACCGAA ACTGCTGTAT TGCAGCAACG GCGGCCATTT TCTGCGCATT 121 CTGCCGGATGGCACCGTGGA TGGCACCCGC GATCGCAGCG ATCAGCATAT TCAGCTGCAG 181 CTGACCGCGGAAAACGTGGG CGAAGTGTAT ATTAAAAGCA CCGAAACCGG CCAGTATCTG 241 GCGATGGATGCGGATGGCCT GCTGTATGGC AGCCAGACCC CGAACGAAGA ATGCCTGTTT 301 CTGGAACGCCTGGAAGAAAA CCATTATAAC ACCTATATTA GCAAAAAACA TGCGGAAAAA 361 AACTGGTTTGTGGCGCTGAA AAAAAACGGC AGCTGCAAAC TGGGCCCGCG CACCCATTAT 421 GGCCAGAAAGCGATTCTGTT TCTGCCGCTG CCGGTGAGCA GCGAT Rock pigeon FGF1 gene codingsequence (1-155) (SEQ ID NO: 90) (generated using SMS Reverse Translatetool on the ExPASy Bioinformatics Resource website (www.expasy.org): 1ATGGCGGAAG GCGAAATTAC CACCTTTACC GCGCTGACCG AAAAATTTAA CCTGCCGCCG 61GGCAACTATA AAAAACCGAA ACTGCTGTAT TGCAGCAACG GCGGCCATTT TCTGCGCATT 121CTGCCGGATG GCAAAGTGGA TGGCACCCGC GATCGCAGCG ATCAGCATAT TCAGCTGCAG 181CTGAGCGCGG AAAGCGTGGG CGAAGTGTAT ATTAAAAGCA CCCAGAGCGG CCAGTATCTG 241GCGATGGATC CGACCGGCCT GCTGTATGGC AGCCAGCTGC TGGGCGAAGA ATGCCTGTTT 301CTGGAACGCA TTGAAGAAAA CCATTATAAC ACCTATGTGA GCAAAAAACA TGCGGATAAA 361AACTGGTTTG TGGGCCTGAA AAAAAACGGC AACAGCAAAC TGGGCCCGCG CACCCATTAT 421GGCCAGAAAG CGATTCTGTT TCTGCCGCTG CCGGTGAGCG CGGAT Sheep FGF1 gene codingsequence (1-155) (SEQ ID NO: 91) (GenBank accession no. XM_004008909,which is hereby incorporated by reference in its entirety): 361ATGGCTGAAG GAGAAACCAC AACCTTCAGG GCCCTGACTG AGAAGTTTAA CCTGCCTCTA 421GGCAATTACA AGAAGCCCAA GCTCCTCTAT TGCAGCAACG GGGGCTACTT CCTGAGAATC 481CTCCCAGATG GCAGAGTGGA TGGGACGAAG GACAGGAGCG ACCAGCACAT TCAGCTGCAG 541CTCTATGCGG AAAGCATAGG GGAGGTGTAT ATTAAGAGTA CGGAGACTGG CCAGTTCTTG 601GCCATGGACA CCAACGGGCT TTTGTACGGC TCACAAACAC CCAGTGAGGA ATGTTTGTTC 661CTGGAAAGGC TGGAGGAAAA CCATTATAAC ACCTACATAT CCAAGAAGCA TGCAGAGAAG 721AATTGGTTCA TTGGTCTCAA GAAGAACGGA AGCTCCAAAC TCGGTCCTCG GACTCACTTC 781GGCCAGAAAG CCATCTTGTT TCTCCCCCTG CCAGTTTCCT CTGATTAA Chicken FGF1 genecoding sequence (1-155) (SEQ ID NO: 92) (GenBank accession no.NM_205180, which is hereby incorporated by reference in its entirety):52                                                         ATGGCCGAG 61GGGGAGATAA CCACCTTCAC CGCCCTGACC GAGCGCTTCG GCCTGCCGCT GGGCAACTAC 121AAGAAGCCCA AACTCCTGTA CTGCAGCAAC GGGGGCCACT TCCTACGGAT CCTGCCGGAC 181GGCAAGGTGG ACGGGACGCG GGACCGGAGT GACCAGCACA TTCAGCTGCA GCTCAGCGCG 241GAAGATGTGG GCGAGGTCTA TATAAAGAGC ACAGCGTCGG GGCAGTACCT GGCAATGGAC 301ACCAACGGGC TCCTGTATGG CTCGCAGCTA CCAGGCGAGG AGTGCTTGTT CCTTGAGAGG 361CTCGAGGAGA ACCATTACAA CACATACATC TCCAAAAAGC ACGCAGACAA GAACTGGTTC 421GTCGGGCTGA AGAAAAACGG GAACAGCAAG CTGGGGCCGC GGACTCACTA TGGGCAAAAG 481GCGATCCTCT TCCTCCCATT GCCGGTGTCG GCTGACTGA Alpaca FGF1 gene codingsequence (1-155, excluding 1-57) (SEQ ID NO: 93) (Ensembl accession no.ENSVPAT00000008395, which is hereby incorporated by reference in itsentirety): 1 CAGCTGCAGC TCAGTGCGGA AAGCGTGGGG GAGGTGTATA TAAAGAGTACCGAGACTGGC 61 CAGTACTTGG CCATGGACAC CGACGGGCTT TTGCACGGCT CACAGACACCAAATGAGGAA 121 TGTTTGTTCC TGGAAAGGCT GGAGGAGAAC CATTACAACA CCTACACGTCCAAGAAGCAC 181 GCCGAAAAGA ATTGGTTTGT TGGTCTCAAG AAGAATGGAA GCTGCAAACGCGGTCCTCGG 241 ACTCACTACG GCCAGAAGGC GATCTTGTTT CTCCCCTTGC CAGTCTCCTCTGATTAA Anole lizard FGF1 gene coding sequence (1-155) (SEQ ID NO: 94)(Ensembl accession no. ENSACAT00000013467, which is hereby incorporatedby reference in its entirety): 1 ATGGCTGAAG GTGAAATAAC AACATTCACAGCCTTGACCG AGAGGTTTGC TCTCCCAATG 61 GAGAATTACA AGAAGCCCAA ACTCCTGTATTGCAGCAATG GAGGCCACTT CCTGAGGATC 121 CTTCCAGATG GAAAAGTGGA TGGCACCATGGACCGGAATG ACAGCTATAT TCAGTTGCTG 181 TTAACAGCAG AAGATGTGGG TGTGGTATATATAAAAGGCA CTGAGACCGG GCAGTACTTG 241 GCCATGGATG CCAATGGACA TTTATATGGCTCGCAGTTGC CAACAGAAGA GTGTTTATTT 301 GTGGAAACGC TGGAAGAAAA CCATTACAATACATATACCT CAAAGATGCA TGGCGATAAG 361 AAGTGGTATG TTGGCTTGAA AAAGAATGGGAAAGGCAAAC TGGGGCCACG GACTCATCGC 421 GGCCAAAAGG CAATACTTTT CCTTCCACTGCCAGTATCAC CTGATTAG Bushbaby FGF1 gene coding sequence (1-155) (SEQ IDNO: 95) (Ensembl accession no. ENSOGAT00000005081, which is herebyincorporated by reference in its entirety): 1 ATGGCTGAAG GGGAAATCACAACCTTCACA GCCCTCACAG AGAAGTTTAA TCTGCCTCTA 61 GGAAATTACA AGAAGCCCAAGCTCCTCTAC TGTAGCAACG GGGGTCACTT TCTGAGGATC 121 CTGCCGGATG GCACCGTGGATGGGACACAA GACAGGAGCG ACCAGCACAT TCAGCTGCAG 181 CTCAGTGCGG AAAGCGTGGGGGAGGTGTAT ATAAAGAGTA CCCAGACTGG CCAGTACTTG 241 GCCATGGACT CCGACGGGCTTTTATACGGC TCACAAACAC CAAATGAGGA ATGCCTGTTC 301 CTGGAACGGC TGGAGGAAAACCATTACAAC ACCTATGTGT CCAAGAAGCA CGCCGAGAAG 361 AATTGGTTTG TCGGTCTCAAGAAGAACGGA AGTTGCAAAC GTGGTCCTCG GACTCACTAC 421 GGCCAGAAAG CAATCTTGTTTCTCCCCCTG CCAGTCTCCT CTGATTAA Cat FGF1 gene coding sequence (1-155)(SEQ ID NO: 96) (Ensembl accession no. ENSFCAT00000009123, which ishereby incorporated by reference in its entirety): 1 ATGGCTGAAGGGGAAATCAC AACCTTCACG GCCCTGACGG AGAAGTTCAA TCTGCCTCCA 61 GGGAATTACAAGAAACCCAA ACTCCTCTAC TGTAGCAACG GGGGCCACTT CCTGAGGATC 121 CTTCCAGATGGCACAGTGGA TGGGACGAGG GACAGGAGCG ACCAGCACAT TCAGCTGCAG 181 CTCAGTGCGGAAAGCGTGGG GGAGGTGTAT ATAAAGAGTA CCGAGACTGG CCAGTACTTG 241 GCCATGGACACCGACGGGCT TTTGTACGGC TCACAGACAC CAAATGAGGA ATGCTTGTTC 301 CTGGAAAGGCTGGAAGAAAA CCATTACAAC ACCTACACAT CCAAGAAGCA CGCAGAAAAG 361 AATTGGTTTGTGGGTCTCAA GAAGAATGGA AGCTGCAAAC GCGGTCCCCG GACTCACTAT 421 GGCCAGAAGGCAATTTTGTT TCTCCCCCTG CCAGTCTCCT CTGATTAA Chinese softshell turtle FGF1gene coding sequence (1-155) (SEQ ID NO: 97) (Ensembl accession no.ENSPSIT00000016432, which is hereby incorporated by reference in itsentirety): 131            ATGGCTGAAG GGGAAATAAC AACGTTCACC GCCCTGACCGAAAAATTCAA 181 CCTTCCCCTG GGGAATTACA AGAATCCCAA ACTCTTATAT TGCAGCAATGGAGGCTACTT 241 CTTGAGGATA CATCCAGATG GCAAAGTAGA TGGGACAAGG GACCGAAGTGACCAACACAT 301 TCAGCTGCAG CTAAGTGCGG AAAGCGTGGG TGAGGTATAT ATAAAGAGCACTGAGTCTGG 361 ACAGTTTTTG GCTATGGACG CCAATGGACT TTTATATGGA TCACTGTCACCGAGTGAGGA 291 ATGCTTATTC TTGGAAAGAA TGGAAGAAAA TCATTATAAC ACCTACATCTCCAAGAAGCA 351 TGCAGACAAG AACTGGTTCG TTGGCTTAAA GAAGAATGGA AGCTGCAAACTGGGACCGCG 411 GACGCACTAC GGCCAAAAGG CCGTCCTTTT CCTTCCACTG CCAGTGTCAGCTGATTAA Coelacanth FGF1 gene coding sequence (1-155) (SEQ ID NO: 98)(Ensembl accession no. ENSLACT00000015212, which is hereby incorporatedby reference in its entirety): 1 ATGGCTGAAG ACAAAATAAC AACACTGAAGGCCTTGGCTG AAAAATTTAA CCTTCCTATG 61 GGAAATTACA AGAAAGCAAA ACTCCTCTACTGCAGCAACG GAGGGTATTT CCTGCGAATA 121 CCCCCAGACG GGAAAGTGGA AGGAATTAGAGAACGAAGCG ACAAGTACAT TCAGCTGCAA 181 ATGAATGCAG AAAGTTTAGG CATGGTGTCTATAAAGGGTG TGGAGGCAGG GCAATACCTA 241 GCTATGAATA CAAATGGACT CCTGTATGGATCTCAGTCTC TAACTGAAGA ATGCCTTTTC 301 ATGGAAAAGA TGGAAGAAAA CCACTACAACACATACAGGT CTAAGACACA TGCAGATAAA 361 AACTGGTATG TTGGCATTAG AAAGAACGGTAGCATCAAAC CAGGACCAAG GACTCACATT 421 GGCCAAAAGG CTGTTCTTTT TCTCCCTCTGCCTGCCTCGA GTGATTAG Dolphin FGF1 gene coding sequence (1-155) (SEQ IDNO: 99) (Ensembl accession no. ENSTTRT00000004742, which is herebyincorporated by reference in its entirety): 1 ATGGCTGAAG GGGAAATCACAACCTTCACA GCCCTGACCG AGAAGTTTAA TCTGCCTCCA 61 GGGAATTACA AGAAGCCCAAACTCCTCTAC TGTAGCAACG GGGGCCACTT CCTGAGGATC 121 CTTCCAGATG GCACAGTGGATGGGACAAGG GACAGGAGTG ACCAGCACAT TCAGCTGCAG 181 CTCAGTGCGG AAAGCGTGGGGGAGGTGTAT ATAAAGAGTA CGGAGACTGG CCAGTACTTG 241 GCCATGGACA CCGACGGGCTTTTGTACGGC TCACAGACAC CCAATGAGGA ATGTTTGTTC 301 CTGGAAAGGT TGGAGGAAAACCATTACAAC ACCTACGCAT CCAAGAAGCA TGCAGAAAAG 361 AATTGGTTCG TTGGTCTCAAGAAGAACGGA AGCTGCAAAC GCGGTCCTCG GACTCACTAC 421 GGCCAGAAAG CAATCTTGTTTCTCCCCCTG CCAGTCTCCT CCGATTAA Ferret FGF1 gene coding sequence (1-155)(SEQ ID NO: 100) (Ensembl accession no. ENSMPUT00000008013, which ishereby incorporated by reference in its entirety): 1                                     ATGGCT GAAGGGGAAA TCACAACCTT 61CACAGCCCTG ATGGAGAAGT TTAATCTGCC TGCGGGGAAT TACAAGAAGC CCAAACTCCT 121CTACTGTAGC AATGGGGGCC ACTTCCTGAG GATCCTTCCA GATGGCACAG TGGACGGCAC 181AAGGGACAGG AGCGACCAGC ACATTCAGCT GCAGCTCAGT GCGGAAAGCG TGGGGGAGGT 241GTACATAAAG AGTACCGAGA CTGGCCAGTA CTTGGCCATG GACACCGATG GGCTTTTGTA 301CGGCTCACAA ACACCAAATG AGGAATGTCT GTTCCTGGAA AGGCTGGAGG AAAACCATTA 361CAACACCTAC ACATCCAAGA AGCACGCTGA GAAGAATTGG TTTGTAGGTC TCAAGAAGAA 421CGGAAGCTGC AAACGCGGTC CTCGGACTCA CTATGGCCAG AAAGCAATTC TGTTTCTCCC 481CCTGCCAGTC TCCTCTGATT AA Gibbon FGF1 gene coding sequence (1-155) (SEQID NO: 101) (Ensembl accession no. ENSNLET00000012455, which is herebyincorporated by reference in its entirety): 241                                                  ATGG CCGAAGGGGA 301AATCACCACC TTCACAGCCC TGACCGAGAA GTTTAATCTG CCTCCAGGGA ATTACAAGAA 361GCCCAAACTC CTCTACTGTA GCAACGGGGG CCACTTCTTG AGGATCCTTC CGGATGGCAC 421AGTGGATGGG ACAAGGGACA GGAGCGACCA GCACATTCAG CTGCAGCTCA GTGCGGAAAG 481CGTGGGGGAG GTGTATATAA AGAGTACCGA GACTGGCCAG TACTTGGCCA TGGACACCGA 541CGGGCTTTTA TACGGCTCAC AGACACCAAA TGAGGAATGT TTGTTCCTGG AAAGGCTGGA 601GGAGAACCAT TACAACACCT ATATATCCAA GAAGCATGCA GAGAAGAATT GGTTTGTTGG 661CCTCAAGAAG AATGGAAGCT GCAAACGCGG TCCTCGGACT CACTATGGCC AGAAAGCAAT 721CTTGTTTCTC CCCCTGCCAG TCTCTTCTGA TTAA Gorilla FGF1 gene coding sequence(1-155) (SEQ ID NO: 102) (Ensembl accession no. ENSGGOT00000025344,which is hereby incorporated by reference in its entirety): 121                                                  ATGG CTGAAGGGGA 181AATCACCACC TTCACAGCCC TGACCGAGAA GTTTAATCTG CCTCCAGGGA ATTACAAGAA 241GCCCAAACTC CTCTACTGTA GCAATGGGGG CCACTTCTTG AGGATCCTTC CGGATGGCAC 301AGTGGATGGG ACAAGGGACA GGAGCGACCA GCACATTCAG CTGCAGCTCA GTGCGGAAAG 361CGTGGGGGAG GTGTATATAA AGAGTACCGA GACTGGCCAG TACTTGGCCA TGGACACCGA 421CGGGCTTTTA TACGGCTCAC AGACACCAAA TGAGGAATGT TTGTTCCTGG AAAGGCTGGA 481GGAGAACCAT TACAACACCT ATATATCCAA GAAGCATGCA GAGAAGAATT GGTTTGTTGG 541CCTCAAGAAG AATGGAAGCT GCAAACGCGG TCCTCGGACT CACTATGGCC AGAAAGCAAT 601CTTGTTTCTC CCCCTGCCAG TCTCTTCCGA TTAA Hedgehog FGF1 gene coding sequence(1-155) (SEQ ID NO: 103) (Ensembl accession no. ENSEEUT00000005832,which is hereby incorporated by reference in its entirety): 1 ATGGCTGAAGGAGAAATCAC CACCTTCACG GCCCTGACTG AGAAGTTTAA TCTGCCACTA 61 GGGAATTACAAGAAGCCCAA GCTCCTCTAC TGTAGCAACG GGGGCCACTT CCTGAGGATC 121 CTTCCAGATGGCACCGTGGA TGGGACAAGG GACAGGAGCG ACCAGCATAT TCAGCTGCAG 181 CTCAGTGCGGAAAGCGTGGG GGAGGTGTAT ATAAAGAGTA CGGAGACTGG CCAGTACTTG 241 GCCATGGACACCGACGGGCT TTTATACGGC TCACAAACAC CAAATGAGGA ATGTCTGTTC 301 CTTGAAAGGCTGGAAGAGAA CCATTACAAT ACCTACACAT CCAAGAAGCA TGCCGAGAAG 361 AACTGGTTTGTTGGCCTCAA GAAGAATGGA AGCTGCAAGC GTGGTCCTCG GACTCATTAT 421 GGCCAGAAAGCTATTTTGTT TCTCCCCCTG CCAGTTTCCT CTGATTAA Hyrax FGF1 gene codingsequence (1-155, excluding 1-90) (SEQ ID NO: 104) (Ensembl accession no.ENSPCAT00000011746, which is hereby incorporated by reference in itsentirety): 1 ATGGCTGAAG GCGAAATCAC AACCTTCACA GCCCTGACTG AGAAGTTTAACCTGCCACTA 61 GAGAATTACA AGAAGCCCAA ACTCCTCTAC TGTAGCAACG GAGGCCACTTCCTGAGGATC 121 CTTCCGGACG GCACAGTGGA TGGCACCAGG GACAGGAGTG ACCAGCACATTCAGCTGCAG 181 CTCAGTGCGG AAAGCGTGGG GGAGGTGTAT ATAAAGGGCA CCGAGACTGGCCAGTACTTG 241 GCCATGGACA CCGACGGGCT TTTATATGGC TCA Kangaroo rat FGF1gene coding sequence (1-155, excluding 1-16 and 58-155) (SEQ ID NO: 105)(Ensembl accession no. ENSDORT00000007345, which is hereby incorporatedby reference in its entirety): 1 ATGGCTGAAG GGGAAATCAC AACCTTCACAGCCCTGACGG AAAGGTTTAA ---------- ---------- ---------- -------------------- ---------- ---------- 51 ---------- ---------- -------------------- ---------T TCAGCTGCAA 62 CTGAGTGCGG AAAGCGTGGG GGAGGTCTATATAAAGAGCA CCGAGACTGG CCAATACTTG 122 GCCATGGATG CCGACGGGCT TTTATACGGCTCACAGACAC CTGATGAAGA ATGCTTGTTC 182 CTGGAGAGGC TGGAAGAAAA TCATTATAACACCTACATAG CCAAGAAACA TGCTGAAAAG 242 AATTGGTTTG TCGGCCTCAA AAAGAATGGAAGCTGCAAGC GTGGTCCTCG GACTCACTAT 302 GGCCAGAAAG CAATCCTGTT CCTCCCCTTGCCTGTCTCCT CTGATTAG Lamprey FGF1 gene coding sequence (1-155, excluding94-155) (SEQ ID NO: 106) (Ensembl accession no. ENSPMAT00000010729,which is hereby incorporated by reference in its entirety): 1 ATGGAGGTGGGCCACATCGG CACGCTGCCC GTGGTCCCCG CGGGGCCCGT GTTCCCCGGC 61 AGTTTCAAGGAGCCACGGCG CCTCTACTGC CGCAGCGCGG GCCACCACCT CCAGATCCTG 121 GGGGACGGCACCGTGAGTGG CACCCAGGAC GAGAACGAGC CCCACGCCGT TCTGCAGCTG 181 CAGGCGGTGCGCCGCGGGGT GGTGACGATC CGTGGGCTCT GCGCCGAGAG GTTCCTCGCC 241 ATGAGCACGGAGGGACACCT GTACGGGGCG GTGAGG Lesser hedgehog tenrec FGF1 gene codingsequence (1-155, excluding 1-57) (SEQ ID NO: 107) (Ensembl accession no.ENSETET00000017851, which is hereby incorporated by reference in itsentirety): 1 CAGCTGAAGC TCGTTGCCGA AAGCGTGGGG GTGGTGTATA TAAAGAGCATCAAGACCGGC 61 CAGTACTTGG CCATGAACCC CGACGGGCTT TTATACGGCT CCGAGACCCCAGAGGAAGAA 121 TGCTTGTTCC TGGAAACGCT GGAGGAAAAC CACTACACCA CCTTCAAATCTAAGAAGCAC 181 GTAGAGAAGA ATTGGTTCGT TGGTCTCCGG AAGAATGGAA GGGTCAAGATCGGGCCTCGG 241 ACTCACCAAG GCCAGAAAGC AATCTTGTTC CTGCCCCTCC CGGTGTCCTCTGATTAA Rhesus monkey FGF1 gene coding sequence (1-155) (SEQ ID NO: 108)(Ensembl accession no. ENSMMUT00000033070, which is hereby incorporatedby reference in its entirety): 36                                      ATGGC TGAAGGGGAA ATCACCACGT 61TCACAGCCCT GACCGAGAAG TTTAATCTGC CTCCAGGGAA TTACAAGAAG CCCAAACTGC 121TCTACTGTAG CAATGGGGGC CACTTCTTGA GGATCCTTCC GGATGGCACA GTGGATGGGA 181CAAGGGACAG GAGCGACCAG CACATTCAGC TGCAGCTCAG TGCGGAAAGC GTGGGGGAGG 241TGTATATAAA GAGTACCGAG ACTGGCCAGT ACTTGGCCAT GGACACCGAC GGGCTTTTAT 301ACGGCTCACA GACACCAAAT GAGGAATGTT TGTTCCTGGA AAGGCTGGAG GAGAACCATT 361ACAACACCTA TACATCCAAG AAGCACGCAG AGAAGAATTG GTTTGTTGGC CTCAAGAAGA 421ATGGAAGCTG CAAACGTGGT CCTCGGACTC ACTATGGCCA GAAAGCAATC TTGTTTCTTC 481CCCTGCCAGT CTCTTCTGAT TAA Megabat FGF1 gene coding sequence (1-155) (SEQID NO: 109) (Ensembl accession no. ENSPVAT00000004596, which is herebyincorporated by reference in its entirety): 1 ATGGCCGAGG GGGAAGTCACGACGTTCACG GCCCTGACCG AGAGGTTTAA CCTGCCTCCA 61 GGGAATTACA AGAAGCCCAAACTTCTCTAC TGCAGCAACG GGGGCCACTT CCTGAGGATC 121 CTCCCAGATG GCACAGTGGATGGGACAAGG GACAAGAGCG ACCAGCACAT TCAGCTGCAG 181 CTCAGTGCGG AAAGTGTGGGGGAGGTGTAT ATAAAGAGCA CCGAGAGTGG CCAGTACTTG 241 GCCATGGACT CCGACGGGCTTTTGTACGGC TCACAGACAC CAGATGAGGA CTGTTTGTTC 301 CTGGAAAGGC TGGAGGAAAACCATTACAAC ACCTACACAT CCAAGAAGCA CGCAGAGAAG 361 AATTGGTTTG TTGGGCTCAAGAAGAATGGA AGCTGCAAGC GCGGTCCCCG GACTCACTAC 421 GGCCAGAAAG CGATCCTGTTTCTCCCCCTG CCAGTCTCCT CTGATTAG Microbat FGF1 gene coding sequence(1-155) (SEQ ID NO: 110) (Ensembl accession no. ENSMLUT00000007098,which is hereby incorporated by reference in its entirety): 66     ATGGC TGAGGGGGAA GTCACCACAT TCACGGCCCT GACCGAGAGG TTCAATCTGC 121CTCTGGAGAA CTACAAGAAG CCCAAGCTTC TCTACTGCAG CAACGGGGGC CACTTCCTGC 181GGATCCTCCC AGACGGCACC GTGGACGGGA CGAGGGACAG GAGCGACCAG CACATTCAGC 241TGCAGCTCAG TGCGGAAAGC GTGGGGGAGG TGTATATAAA GAGCACCGAG AGTGGCCAGT 301ACTTGGCCAT GGACTCCGAC GGGCTTTTGT ACGGCTCACA AACACCCAAT GAGGAATGTT 361TGTTCCTGGA AAGGCTGGAG GAGAACCACT ACAACACCTA CACGTCCAAG AAGCACGCAG 421AAAAGAATTG GTTCGTTGGG CTCAAGAAGA ACGGAAGCTG CAAGCGTGGT CCTCGGACGC 481ATTATGGCCA GAAAGCAATC TTGTTTCTCC CCCTGCCAGT CTCCTCCGAT TAA Mouse lemurFGF1 gene coding sequence (1-155) (SEQ ID NO: 111) (Ensembl accessionno. ENSMICT00000009454, which is hereby incorporated by reference in itsentirety): 1 ATGGCCGAAG GGGAGATCAC AACCTTCACG GCCCTCACCG AGAAGTTTAACCTGCCTCCG 61 GGGAACTACA AGAAGCCCAA GCTCCTCTAC TGCAGCAACG GCGGCCACTTCCTGCGCATC 121 CTTCCCGACG GCACCGTGGA TGGCACGAGA GACAGGAGCG ACCAGCACATTCAGCTGCAG 181 CTCAGTGCGG AAAGCGCGGG GGAGGTGTAT ATAAAGAGCA CCCAGACTGGCCGGTACTTG 241 GCCATGGACG CCGACGGGCT TTTATACGGC TCACAAACAC CAAATGAGGAATGTTTGTTC 301 CTGGAAAGGC TGGAGGAAAA CCATTACAAC ACCTACGTAT CCAAGAAGCACGCAGAGAAG 361 AATTGGTTTG TTGGCCTCAA GAAGAATGGA AGTTGCAAAC GCGGCCCCCGGACTCACTAT 421 GGCCAGAAAG CAATCTTGTT TCTGCCCCTG CCAGTCTCCT CTGATTAA PikaFGF1 gene coding sequence (1-155, excluding 57-67) (SEQ ID NO: 112)(Ensembl accession no. ENSOPRT00000012854, which is hereby incorporatedby reference in its entirety): 1 ATGGCCGAGG GAGAAGTCAC CACCTTCTCAGCCCTGACGG AGAAGTTCAA TCTGCCTGGA 61 GGAAACTACA AGTTGCCCAA GCTCCTTTACTGTAGCAACG GAGGCCACTT CCTGAGGATC 121 CTTCCAGATG GCACAGTGGA TGGGACCAGGGACAGGAGCG ACCTGCACA- ---------- 170 ---------- ---------- -GAGGTGTTTATAAAGAGTA CGGAGACTGG CCAGTACTTG 209 GCTATGGACA CCGATGGCCT TTTATATGGCTCGCAGACAC CCAGTGAGGA GTGTTTGTTC 269 CTGGAGCGGC TGGAGGAGAA CCACTACAACACCTACACAT CCAAGAAGCA TGCCGAGAAG 329 AACTGGTTTG TGGGCATCAA GAAGAATGGAAGCTGCAAGC GTGGTCCTCG GACTCACTAC 389 GGCCAGAAAG CCATCTTGTT TCTCCCTCTGCCAGTCTCTT CTGACTAA Rat FGF1 gene coding sequence (1-155) (SEQ ID NO:113) (Ensembl accession no. ENSRNOT00000018577, which is herebyincorporated by reference in its entirety): 268                             ATG GCCGAAGGGG AGATCACAAC CTTTGCAGCC 301CTGACCGAGA GGTTCAATCT GCCTCTAGGG AACTACAAAA AACCCAAACT GCTCTACTGC 361AGCAACGGGG GCCACTTCTT GAGGATTCTT CCCGATGGCA CCGTGGATGG GACCAGGGAC 421AGGAGCGACC AGCACATTCA GCTGCAGCTC AGTGCGGAAA GCGCGGGCGA AGTGTATATA 481AAGGGTACAG AGACTGGCCA GTACTTGGCC ATGGACACCG AAGGGCTTTT ATACGGCTCG 541CAGACACCAA ATGAAGAATG CCTATTCCTG GAAAGGCTAG AAGAAAACCA TTATAACACT 601TACACATCCA AGAAGCACGC GGAGAAGAAC TGGTTTGTGG GCCTCAAGAA GAACGGGAGT 661TGTAAGCGCG GTCCTCGGAC TCACTACGGC CAGAAAGCCA TCTTGTTTCT CCCCCTCCCG 721GTATCTTCTG ACTAA Sloth FGF1 gene coding sequence (1-155) (SEQ ID NO:114) (Ensembl accession no. ENSCHOT00000012416, which is herebyincorporated by reference in its entirety): 1 ATGGCTGAAG GGGAAATCACAACCTTCACA GCTCTGATGG AGAAGTTTAA CCTGCCACCA 61 GGGAATTACA TGAAGCCCAAACTCCTCTAC TGTAGCAACG GGGGCCACTT CTTGAGGATC 121 CTTCCAGACG GCACAGTGGATGGGACAAGG GACAGGAGCG ACCTGCACAT TCAGCTGCAG 181 CTCAGTGCGG AAAGCGTGGGGGAGGTGTAT ATAAAGAGTG CGGAGACCGG CCAGTACTTA 241 GCCATGGACA CCGGCGGGCTTTTATACGGC TCACAGACAC CAAGTGAGGA ATGCCTGTTC 301 CTAGAAAGGC TGGAGGAAAACCATTACAAC ACCTACGTAT CCAAGAAGCA TGCGGAGAAG 361 AACTGGTTCG TTGGCCTAAAGAAGAATGGA AGCAGCAAAC GCGGCCCCCG GACTCACTAT 421 GGCCAGAAAG CCATCTTGTTTCTTCCCCTG CCAGTCTCCT CTGATTAA Squirrel FGF1 gene coding sequence(1-155) (SEQ ID NO: 115) (Ensembl accession no. ENSSTOT00000029249,which is hereby incorporated by reference in its entirety): 1                                                             ATGG 5CTGAAGGGGA AATCACAACC TTCACAGCCC TGACCGAGAA GTTCAATCTG CCTCCAGGGA 65ACTACAAGAA GCCCAAACTG CTCTACTGTA GCAACGGAGG CCACTTCTTG AGGATCCTTC 125CTGATGGCAC AGTGGATGGG ACAAGAGACA GGAGCGACCA ACACATTCAG CTGCAGCTCA 185GTGCGGAAAG CGTGGGGGAG GTGTATATAA AGAGTACCGA GACCGGCCAG TACTTGGCCA 245TGGACACCGA CGGGCTTTTA TATGGCTCAC AGACCCCAAA TGAGGAATGC TTATTCCTGG 305AAAGGCTGGA GGAAAACCAT TACAACACGT ACACATCCAA GAAGCATGCA GAGAAGAATT 365GGTTTGTTGG CCTCAAGAAG AACGGAAGCT GCAAGCGCGG TCCCCGGACT CACTATGGCC 425AGAAAGCGAT CTTGTTTCTC CCACTGCCTG TCTCCTCTGA TTAG Tarsier FGF1 genecoding sequence (1-155) (SEQ ID NO: 116) (Ensembl accession no.ENSTSYT00000007425, which is hereby incorporated by reference in itsentirety): 1 ATGGCCGAAG GGGAAATCAC AACCTTCACA GCCCTGACCG AGAAGTTCAACCTGCCCCCG 61 GGGAATTACA AGAAGCCCAA ACTCCTCTAC TGCAGCAACG GGGGCCACTTCTTGAGGATC 121 CTTCCGGATG GCACTGTGGA TGGAACGAGG GACAGGAGCG ACCAGCACATTCAGCTGCAG 181 CTCAGCGCGG AAAGCGTGGG GGAGGTGTAT ATAAAGAGTA CCGAGACCGGCCAGTACTTG 241 GCCATGGACA CCGACGGGCT TTTGTACGGC TCACAGACAC CAAATGAGGAGTGTCTGTTC 301 CTGGAAAGGC TGGAAGAGAA TCATTACAAT ACCTACGTGT CCAAGAAGCATGCGGAGAAG 361 AATTGGTTTG TCGGCCTCAA GAAGAATGGA AGCTGCAAAC GCGGTCCTCGGACTCACTAT 421 GGCCAGAAAG CAATCTTGTT TCTCCCCCTG CCAGTTTCCT CTGATTAA Treeshrew FGF1 gene coding sequence (1-155) (SEQ ID NO: 117) (Ensemblaccession no. ENSTBET00000011861, which is hereby incorporated byreference in its entirety): 1 ATGGCTGAAG GGGAAATCAC GACCTTCGCAGCCCTGACCG AGAAGTTTGA TCTGCCTCCA 61 GGGAATTACA AGAAGCCCAA ACTTCTCTACTGTAGCAACG GGGGCCATTT CTTGAGGATT 121 CTTCCAGATG GCACCGTGGA TGGGACAAGAGACAGGAGCG ACCAGCACAT TCAGCTGCAG 181 CTCACTGCGG AAAACGTGGG GGAGGTGTACATAAAGAGTA CGGAGACTGG CCAGTACTTG 241 GCCATGGACG CCGACGGGCT TTTATATGGCTCACAGACAC CAAACGAGGA ATGTTTGTTC 301 CTGGAAAGGC TGGAGGAGAA CCATTACAACACCTACATAT CCAAGAAGCA CGCAGAGAAG 361 AATTGGTTTG TTGCCCTCAA GAAGAACGGAAGCTGCAAAC TCGGTCCTCG GACTCACTAT 421 GGCCAGAAAG CAATCTTGTT TCTCCCCCTGCCAGTCTCCT CTGATTAA Turkey FGF1 gene coding sequence (1-155, excluding57-155) (SEQ ID NO: 118) (Ensembl accession no. ENSMGAT00000017372,which is hereby incorporated by reference in its entirety): 1 ATGGCCGAGGGGGAGATAAC CACCTTCACA GCCCTGACCG AGCGCTTCGG CCTGCCGCTG 61 GGCAACTACAAGAAGCCCAA ACTCCTGTAC TGCAGCAACG GGGGCCACTT CCTACGGATC 121 CTGCCGGACGGCAAGGTGGA CGGGACGCGG GACCGGAGCG ACCAGCAC Wallaby FGF1 gene codingsequence (1-155) (SEQ ID NO: 119) (Ensembl accession no.ENSMEUT00000016544, which is hereby incorporated by reference in itsentirety): 1 ATGGCCGAAG GGGAGATCAC AACCTTCACA GCCCTGACCG AAAGATTTAACCTGCCACTG 61 GGGAATTACA AGAAGCCCAA GCTTCTCTAC TGTAGCAATG GGGGCCACTTTTTGAGGATC 121 CTTCCTGATG GCAAAGTGGA TGGGACAAGG GACAGAAATG ATCAACACATTCAACTGCAA 181 CTAAGCGCGG AAAGCGTGGG TGAGGTGTAT ATAAAGAGCA CTGAGTCTGGGCAGTATTTG 241 GCCATGGACA CCAATGGACT TTTATATGGC TCACAGACCC CCAGCGAAGAATGCTTATTC 301 CTGGAGAGGT TGGAGGAGAA TCATTACAAC ACCTACATAT CAAAGAAGCATGCGGAGAAA 361 AATTGGTTTG TTGGCCTCAA GAAGAACGGA AGTTGCAAAA GAGGTCCCAGGACTCACTAT 421 GGCCAGAAAG CCATCCTATT CCTTCCCCTC CCTGTGTCCT CTGAGTAAZebrafish FGF1 gene coding sequence (1-147) (SEQ ID NO: 120) (Ensemblaccession no. ENSDART00000005842, which is hereby incorporated byreference in its entirety): 178                                                              ATG 181ACCGAGGCCG ATATTGCGGT AAAGTCCAGC CCGCGCGACT ATAAAAAACT GACGCGGCTG 241TACTGTATGA ATGGAGGATT TCACCTTCAG ATCCTGGCGG ACGGGACAGT GGCTGGAGCA 124GCAGACGAAA ACACATACAG CATACTGCGC ATAAAAGCAA CAAGTCCAGG AGTGGTGGTG 184ATCGAAGGAT CAGAAACAGG TCTTTACCTC TCGATGAATG AACATGGCAA GCTGTACGCT 244TCATCATTAG TGACGGATGA AAGTTATTTC CTGGAGAAGA TGGAGGAAAA CCACTACAAC 304ACATATCAGT CTCAAAAGCA CGGTGAAAAC TGGTACGTCG GAATAAAAAA GAACGGGAAA 364ATGAAACGGG GCCCAAGAAC TCACATCGGA CAAAAGGCCA TTTTCTTTCT TCCACGACAG 424GTGGAGCAGG AAGAGGACTG A

As noted above, also encompassed within the present invention areportions of paracrine FGFs other than FGF1 (e.g., FGF2, FGF4, FGF5,FGF6, FGF9, FGF16, and FGF20). The portions derived from paracrine FGF2include portions corresponding to the above-identified amino acidsequences of FGF1. Corresponding portions may be determined by, forexample, sequence analysis and structural analysis.

In one embodiment, the paracrine FGF is FGF2. In one embodiment, theportion of the FGF2 is derived from human FGF2 having the amino acidsequence of SEQ ID NO: 121 (GenBank Accession No. EAX05222, which ishereby incorporated by reference in its entirety), as follows:

1 MAAGSITTLP ALPEDGGSGA FPPGHFKDPK RLYCKNGGFF LRIHPDGRVD GVREKSDPHI 61KLQLQAEERG VVSIKGVCAN RYLAMKEDGR LLASKCVTDE CFFFERLESN NYNTYRSRKY 121TSWYVALKRT GQYKLGSKTG PGQKAILFLP MSAKS

In one embodiment, the portion of the paracrine FGF includes an aminoacid sequence beginning at any one of residues 1 to 25 and ending at anyone of residues 151 to 155 of SEQ ID NO: 121. In one embodiment, theportion of the paracrine FGF includes amino acid residues 1-151, 1-152,1-153, 1-154, 1-155, 2-151, 2-152, 2-153, 2-154, 2-155, 3-151, 3-152,3-153, 3-154, 3-155, 4-151, 4-152, 4-153, 4-154, 4-155, 5-151, 5-152,5-153, 5-154, 5-155, 6-151, 6-152, 6-153, 6-154, 6-155, 7-151, 7-152,7-153, 7-154, 7-155, 8-151, 8-152, 8-153, 8-154, 8-155, 9-151, 9-152,9-153, 9-154, 9-155, 10-151, 10-152, 10-153, 10-154, 10-155, 11-151,11-152, 11-153, 11-154, 11-155, 12-151, 12-152, 12-153, 12-154, 12-155,13-151, 13-152, 13-153, 13-154, 13-155, 14-151, 14-152, 14-153, 14-154,14-155, 15-151, 15-152, 15-153, 15-154, 15-155, 16-151, 16-152, 16-153,16-154, 16-155, 17-151, 17-152, 17-153, 17-154, 17-155, 18-151, 18-152,18-153, 18-154, 18-155, 19-151, 19-152, 19-153, 19-154, 19-155, 20-151,20-152, 20-153, 20-154, 21-155, 21-151, 21-152, 21-153, 21-154, 21-155,22-151, 22-152, 22-153, 22-154, 22-155, 23-151, 23-152, 23-153, 23-154,23-155, 24-151, 24-152, 24-153, 24-154, 24-155, 25-151, 25-152, 25-153,25-154, or 25-155 of FGF2 (SEQ ID NO: 121). In one embodiment, theportion of the paracrine FGF includes amino acid residues 1-151 or 1-152of SEQ ID NO: 121.

In one embodiment, the portion of the paracrine FGF of the chimericprotein includes an amino acid sequence that has at least 80%, at least85%, at least 90%, at least 95%, at least 97% or at least 99% amino acidsequence identity to the corresponding amino acid sequence of nativeparacrine FGF (e.g., SEQ ID NO: 121). In one embodiment, the portion ofthe paracrine FGF includes an amino acid sequence that has at least 80%,at least 85%, at least 90%, at least 95%, at least 97% or at least 99%amino acid sequence identity to an amino acid sequence beginning at anyone of residues 1 to 25 and ending at any one of residues 151 to 155 ofSEQ ID NO: 121. In one embodiment, the portion of the paracrine FGFincludes an amino acid sequence that has at least 80%, at least 85%, atleast 90%, at least 95%, at least 97% or at least 99% amino acidsequence homology to the corresponding amino acid sequence of nativeparacrine FGF (e.g., SEQ ID NO: 121). In one embodiment, the portion ofthe paracrine FGF includes an amino acid sequence that has at least 80%,at least 85%, at least 90%, at least 95%, at least 97% or at least 99%amino acid sequence homology to an amino acid sequence beginning at anyone of residues 1 to 25 and ending at any one of residues 151 to 155 ofSEQ ID NO: 121.

Also encompassed within the present invention are portions of paracrineFGFs other than FGF2 (e.g., FGF1, FGF4, FGF5, FGF6, FGF9, FGF16, andFGF20). The portions derived from paracrine FGFs other than FGF2 includeportions corresponding to the above-identified amino acid sequences ofFGF2. Corresponding portions may be determined by, for example, sequenceanalysis and structural analysis.

In one embodiment of the present invention, the portion of the paracrineFGF is derived from an ortholog of a human paracrine FGF. In oneembodiment of the present invention, the portion of the paracrine FGF ofthe chimeric protein is derived from an ortholog of human FGF2. In oneembodiment, the portion of the FGF2 is derived from Gorilla gorilla,Pongo abelii, Macaca mulatta, Pan troglodytes, Pan paniscus, Saimiriboliviensis boliviensis, Nomascus leucogenys, Equus caballus, Bostaurus, Papio Anubis, Vicugna pacos, Ovis aries, Capreolus capreolus,Loxodonta Africana, Sus scrofa, Ailuropoda melanoleuca, Choloepushoffmanni, Bubalus bubalis, Canis lupus familiaris, Rattus norvegicus,Heterocephalus glaber, Otolemur garnettii, Mus musculus, Ictidomystridecemlineatus, Felis catus, Cavia porcellus, Sarcophilus harrisii,Monodelphis domestica, Oryctolagus cuniculus, Meleagris gallopavo,Gallus gallus, Taeniopygia guttata, Cynops pyrrhogaster, Xenopus laevis,Didelphis albiventris, Myotis lucifugus, Anolis carolinensis, Dasypusnovemcinctus, Tupaia belangeri, Xenopus silurana tropicalis, Latimeriachalumnae, Tetraodon nigroviridis, Gasterosteus aculeatus, Takifugurubripes, Oncorhynchus mykiss, Salmo salar, Danio rerio, Oreochromisniloticus, or Oryzias latipes. The portions of an ortholog of humanparacrine FGF include portions corresponding to the above-identifiedamino acid sequences of FGF2. Corresponding portions may be determinedby, for example, sequence analysis and structural analysis.

In one embodiment, the portion of the FGF2 of the chimeric protein ofthe present invention is derived from an ortholog of human FGF2 havingthe amino acid sequence shown in Table 3.

TABLE 3 Amino acid sequence of Gorilla gorilla (gorilla) FGF2 (SEQ IDNO: 122) (Ensembl accession no. ENSGGOP00000004720, which is herebyincorporated by reference in its entirety): 104                                                MAAGSI TTLPALPEDG 120GSGAFPPGHF KDPKRLYCKN GGFFLRIHPD GRVDGVREKS DPHIKLQLQA EERGVVSIKG 180VCANRYLAMK EDGRLLASKC VTDECFFFER LESNNYNTYR SRKYTSWYVA LKRTGQYKLG 240SKTGPGQKAI LFLPMSAKS Amino acid sequence of Pongo abelii (sumatranorangutan) FGF2 (SEQ ID NO: 123) (GenBank accession no. XP_002815172,which is hereby incorporated by reference in its entirety): 168                                                   MAA GSITTLPALP 181EDGGSGAFPP GHFKDPKRLY CKNGGFFLRI HPDGRVDGVR EKSDPHIKLQ LQAEERGVVS 241IKGVCANRYL AMKEDGRLLA SKCVTDECFF FERLESNNYN TYRSRKYTSW YVALKRTGQY 301KLGSKTGPGQ KAILFLPMSA KS Amino acid sequence of Macaca mulatta (rhesusmonkey) FGF2 (SEQ ID NO: 124) (GenBank accession no. XP_001099284, whichis hereby incorporated by reference in its entirety): 83                        MAAGSITT LPALPEDGGS GAFPPGHFKD PKRLYCKNGG 121FFLRIHPDGR VDGVREKSDP HIKLQLQAEE RGVVSIKGVC ANRYLAMKED GRLLASKCVT 181DECFFFERLE SNNYNTYRSR KYTSWYVALK RTGQYKLGSK TGPGQKAILF LPMSAKS Aminoacid sequence of Pan troglodytes (chimpanzee) FGF2 (SEQ ID NO: 125)(GenBank accession no. NP_001103711, which is hereby incorporated byreference in its entirety): 134               MAAGSIT TLPALPEDGGSGAFPPGHFK DPKRLYCKNG GFFLRIHPDG 181 RVDGVREKSD PHIKLQLQAE ERGVVSIKGVCANRYLAMKE DGRLLASKCV TDECFFFERL 241 ESNNYNTYRS RKYTSWYVAL KRTGQYKLGSKTGPGQKAIL FLPMSAKS Amino acid sequence of Pan paniscus (Pygmychimpanzee) FGF2 (SEQ ID NO: 126) (GenBank accession no. XP_003816481,which is hereby incorporated by reference in its entirety): 112                                                        MAAGSITTL 121PALPEDGGSG AFPPGHFKDP KRLYCKNGGF FLRIHPDGRV DGVREKSDPH IKLQLQAEER 181GVVSIKGVCA NRYLAMKEDG RLLASKCVTD ECFFFERLES NNYNTYRSRK YTSWYVALKR 241TGQYKLGSKT GPGQKAILFL PMSAKS Amino acid sequence of Saimiri boliviensisboliviensis (Bolivian squirrel monkey) FGF2 (SEQ ID NO: 127) (GenBankaccession no. XP_003936290, which is hereby incorporated by reference inits entirety): 1 MAAGSITTLP ALPEDGGSGA FPPGHFKDPK RLYCKNGGFF LRIHPDGRVDGVREKSDPHI 61 KLQLQAEERG VVSIKGVCAN RYLAMKEDGR LLASKCVTDE CFFFERLESNNYNTYRSRKY 121 TSWYVALKRT GQYKLGSKTG PGQKAILFLP MSAKS Amino acidsequence of Nomascus leucogenys (Northern white-cheeked gibbon) FGF2(SEQ ID NO: 128) (GenBank accession no. XP_003271404, which is herebyincorporated by reference in its entirety): 1 MAAGSITTLP ALPEDGGSGAFPPGHFKDPK RLYCKNGGFF LRIHPDGRVD GVREKSDPHI 61 KLQLQAEERG VVSIKGVCANRYLAMKEDGR LLASKCVTDE CFFFERLESN NYNTYRSRKY 121 TSWYVALKRT GQYKLGSKTGPGQKAILFLP MSAKS Amino acid sequence of Equus caballus (horse) FGF2 (SEQID NO: 129) (GenBank accession no. NP_001182150, which is herebyincorporated by reference in its entirety): 1 MAAGSITTLP ALPEDGGSGAFPPGHFKDPK RLYCKNGGFF LRIHPDGRVD GVREKSDPHI 61 KLQLQAEERG VVSIKGVCANRYLAMKEDGR LLASKCVTDE CFFFERLESN NYNTYRSRKY 121 SSWYVALKRT GQYKLGPKTGPGQKAILFLP MSAKS Amino acid sequence of Bos taurus (cattle) FGF2 (SEQ IDNO: 130) (GenBank accession no. NP_776481, which is hereby incorporatedby reference in its entirety): 1 MAAGSITTLP ALPEDGGSGA FPPGHFKDPKRLYCKNGGFF LRIHPDGRVD GVREKSDPHI 61 KLQLQAEERG VVSIKGVCAN RYLAMKEDGRLLASKCVTDE CFFFERLESN NYNTYRSRKY 121 SSWYVALKRT GQYKLGPKTG PGQKAILFLPMASKS Amino acid sequence of Papio anubis (Olive baboon) FGF2 (SEQ IDNO: 131) (GenBank accession no. XP_003899210, which is herebyincorporated by reference in its entirety): 1 MAAGSITTLP ALPEDGGSGAFPPGHFKDPK RLYCKNGGFF LRIHPDGRVD GVREKSDPHI 61 KLQLQAEERG VVSIKGVCANRYLAMKEDGR LLASKCVTDE CFFFERLESN NYNTYRSRKY 121 TSWYVALKRT GQYKLGSKTGPGQKAILFLP MSAKS Amino acid sequence of Vicugna pacos (alpaca) FGF2 (SEQID NO: 132) (Ensembl accession no. ENSVPAP00000009804, which is herebyincorporated by reference in its entirety): 111                                                       MAAGSITTLP 121ALPEDGGSGA FPPGHFKDPK RLYCKNGGFF LRIHPDGRVD GVREKSDPHI KLQLQAEERG 181VVSIKGVCAN RYLAMKEDGR LLASKCVTDE CFFFERLESN NYNTYRSRKY SSWYVALKRT 241GQYKLGPKTG PGQKAILFLP MSAKS Amino acid sequence of Ovis aries (sheep)FGF2 (SEQ ID NO: 133) (GenBank accession no. NP_001009769, which ishereby incorporated by reference in its entirety): 1 MAAGSITTLPALPEDGGSSA FPPGHFKDPK RLYCKNGGFF LRIHPDGRVD GVREKSDPHI 61 KLQLQAEERGVVSIKGVCAN RYLAMKEDGR LLASKCVTDE CFFFERLESN NYNTYRSRKY 121 SSWYVALKRTGQYKLGPKTG PGQKAILFLP MSAKS Amino acid sequence of Capreolus capreolus(Western roe deer) FGF2 (partial amino acid sequence corresponding tohuman FGF2 residues 42 to 149) (SEQ ID NO: 134) (GenBank accession no.AAF73226, which is hereby incorporated by reference in its entirety): 1RIHPDGRVDG VREKSDPHIK LQLQAEERGV VSIKGVCANR YLAMKEDGRL LASKCVTDEC 61FFFERLESNN YNTYRSRKYS SWYVALKRTG QYKLGPKTGP GQKAILFL Amino acid sequenceof Loxodonta africana (elephant) FGF2 (partial amino acid sequencecorresponding to human FGF2 residues 60 to 155) (SEQ ID NO: 135)(Ensembl accession no. ENSLAFP00000008249, which is hereby incorporatedby reference in its entirety): 1 VKLQLQAEER GVVSIKGVCA NRYLAMKEDGRLLASRCVTD ECFFFERLES NNYNTYRSRK 61 YTSWYVALKR TGQYKLGSKT GPGQKAILFLPMSAKS Amino acid sequence of Sus scrofa (pig) FGF2 (partial amino acidsequence corresponding to human FGF2 residues 36 to 155) (SEQ ID NO:136) (GenBank accession no. CAE11791 and Ensembl accession no.ENSSSCP00000009695, which is hereby incorporated by reference in itsentirety): 1 NGGFFLRIHP DGRVDGVREK SDPHIKLQLQ AEERGVVSIK GVCANRYLAMKEDGRLLASK 61 CVTDECFFFE RLESNNYNTY RSRKYSSWYV ALKRTGQYKL GPKTGPGQKAILFLPMSAKS Amino acid sequence of Ailuropoda melanoleuca (panda) FGF2(partial amino acid sequence corresponding to human FGF2 residues 60 to155) (SEQ ID NO: 137) (Ensembl accession no. ENSAMEP00000018489, whichis hereby incorporated by reference in its entirety): 1 VKLQLQAEERGVVSIKGVCA NRYLAMKEDG RLLASKCVTD ECFFFERLES NNYNTYRSRK 61 YSSWYVALKRTGQYKLGPKT GPGQKAILFL PMSAKS Amino acid sequence of Choloepus hoffmanni(sloth) FGF2 (SEQ ID NO: 138) (Ensembl accession no. ENSCHOP00000010051,which is hereby incorporated by reference in its entirety): 14                                                          MAAGSIT 21TLPALPEDGG SGALPPGHFK DPKRLYCKNG GFFLRIHPDG RVDGVREKSD PHIKLQLQAE 81ERGVVSIKGV CANRYLAMKE DGRLQASKCV TDECFFFERL ESNNYNTYRS RKYSSWYVAL 141KRTGQYKLGP KTGPGQKAIL FLPMSAKS Amino acid sequence of Bubalus bubalis(water buffalo) FGF2 (SEQ ID NO: 139) (GenBank accession no. AFH66795,which is hereby incorporated by reference in its entirety): 1 MAAGSITTLPPLPEDGGSGA FPPGHFKDPK RLYCKNGGFF LRIHPDGRVD GVREKSDPHI 61 KLQLQAEERGVVSIKGVCAN RYLAMKEDGR LLASKCVTDE CFFFERLESS NYNTYRSRKY 121 SSWYVALKRTGQYKLGPKTG PGQKAILFLP MSAKS Amino acid sequence of Canis lupusfamiliaris (dog) FGF2 (SEQ ID NO: 140) (GenBank accession no.XP_003432529, which is hereby incorporated by reference in itsentirety): 40                                           M AAGSITTLPALPEDGGSGAF 61 PPGHFKDPKR LYCKKGGFFL RIHPDGRVDG VREKSDPHVK LQLQAEERGVVSIKGVCANR 121 YLAMKEDGRL LASKCVTDEC FFFERLESNN YNTYRSRKYS SWYVALKRTGQYKLGPKTGP 181 GQKAILFLPM SAKS Amino acid sequence of Rattus norvegicus(Norway rat) FGF2 (SEQ ID NO: 141) (GenBank accession no. NP_062178,which is hereby incorporated by reference in its entirety): 1 MAAGSITSLPALPEDGGGAF PPGHFKDPKR LYCKNGGFFL RIHPDGRVDG VREKSDPHVK 61 LQLQAEERGVVSIKGVCANR YLAMKEDGRL LASKCVTEEC FFFERLESNN YNTYRSRKYS 121 SWYVALKRTGQYKLGSKTGP GQKAILFLPM SAKS Amino acid sequence of Heterocephalus glaber(naked mole-rat) FGF2 (partial amino acid sequence corresponding tohuman FGF2 residues 22 to 155) (SEQ ID NO: 142) (GenBank accession no.EHB17407, which is hereby incorporated by reference in its entirety): 1ppghfkdpkr lycknggffl rihpdgrvdg vreksdphvk lqlqaeergv vsikgvcanr 61ylamkedgrl laskcvtdec ffferlesnn yntyrsrkys swyvalkrtg qyklgsktgp 121gqkailflpm saks Amino acid sequence of Otolemur garnettii (bushbaby)FGF2 (SEQ ID NO: 143) (Ensembl accession no. ENSOGAP00000021960, whichis hereby incorporated by reference in its entirety): 52                                                        MAAGSITTL 61PSLPEDGGSD AFPPGHFKDP KRLYCKNGGF FLRIHPDGRV DGVREKSDPY IKLQLQAEER 121GVVSIKGVCA NRYLAMKEDG RLLASKLITD ECFFFERLES NNYNTYRSRK YSSWYVALKR 181TGQYKLGSKT GPGQKAILFL PMSAKS Amino acid sequence of Mus musculus (housemouse) FGF2 (SEQ ID NO: 144) (GenBank accession no. NP_032032, which ishereby incorporated by reference in its entirety): 1 MAASGITSLPALPEDGGAAF PPGHFKDPKR LYCKNGGFFL RIHPDGRVDG VREKSDPHVK 61 LQLQAEERGVVSIKGVCANR YLAMKEDGRL LASKCVTEEC FFFERLESNN YNTYRSRKYS 121 SWYVALKRTGQYKLGSKTGP GQKAILFLPM SAKS Amino acid sequence of Ictidomystridecemlineatus (squirrel) FGF2 (partial amino acid sequencecorresponding to human FGF2 residues 12 to 155) (SEQ ID NO: 145)(Ensembl accession no. ENSSTOP00000015653, which is hereby incorporatedby reference in its entirety): 1 LPEDGGGGAF PPGHFKDPKR LYCKNGGFFLRIHPDGRVDG VREKSDPHIK LQLQAEDRGV 61 VSIKGVCANR YLAMKEDGRL LASKCVTDECFFFERLESNN YNTYRSRKYS SWYVALKRTG 121 QYKLGSKTGP GQKAILFLPM SAKS Aminoacid sequence of Felis catus (domestic cat) FGF2 (partial amino acidsequence corresponding to human FGF2 residues 25 to 130) (SEQ ID NO:146) (GenBank accession no. ABY47638, which is hereby incorporated byreference in its entirety): 1 HFKDPKRLYC KNGGFFLRIH PDGRVDGVREKSDPHIKLQL QAEERGVVSI KGVCANRYLA 61 MKEDGRLLAS KCVTDECFFF ERLESNNYNTYRSRKYSSWY VALKRT Amino acid sequence of Cavia porcellus (guinea pig)FGF2 (partial amino acid sequence corresponding to human FGF2 residues60 to 155) (SEQ ID NO: 147) (Ensembl accession no. ENSCPOP00000004847,which is hereby incorporated by reference in its entirety): 1 VKLQLQAEDRGVVSIKGVCA NRYLAMKEDG RLLASKCVTD ECFFFERLES NNYNTYRSRK 61 YSSWYVALKRTGQYKLGSKT GPGQKAILFL PMSAKS Amino acid sequence of Sarcophilus harrisii(Tasmanian devil) FGF2 (SEQ ID NO: 148) (Ensembl accession no.ENSSHAP00000012215, which is hereby incorporated by reference in itsentirety): 48                                                    MAAGSITTLPALA 61 GDGASGGAFP PGHFQDPKRL YCKNGGFFLR IHPDGHVDGI REKSDPHIKLQLQAEERGVV 121 SIKGVCANRY LAMKEDGRLL ALKCVTEECF FFERLESNNY NTYRSRKYSNWYVALKRTGQ 181 YKLGSKTGPG QKAILFLPMS AKS Amino acid sequence ofMonodelphis domestica (gray short-tailed opossum) FGF2 (SEQ ID NO: 149)(GenBank accession no. NP_001029148, which is hereby incorporated byreference in its entirety): 1 MAAGSITTLP ALSGDGGGGG AFPPGHFKDPKRLYCKNGGF FLRIHPDGRV DGIREKSDPN 61 IKLQLQAEER GVVSIKGVCA NRYLAMKEDGRLLALKYVTE ECFFFERLES NNYNTYRSRK 121 YSNWYVALKR TGQYKLGSKT GPGQKAILFLPMSAKS Amino acid sequence of Oryctolagus cuniculus (rabbit) FGF2 (SEQID NO: 150) (GenBank accession no. XP_002717284, which is herebyincorporated by reference in its entirety): 1 MAAESITTLP ALPEDGGSGAFPPGHFKDPK RLYCKNGGFF LRIHPDGRVD GVREKSDPHI 61 KLQLQAEERG VVSIKGVCANRYLAMKEDGR LLASKCVTDE CFFFERLESN NYNTYRSRKY 121 SSWYVALKRT GQYKLGSKTGPGQKAILFLP MSAKS Amino acid sequence of Meleagris gallopavo (turkey)FGF2 (partial amino acid sequence corresponding to human FGF2 residues31 to 155) (SEQ ID NO: 151) (Ensembl accession no. ENSMGAP00000010977,which is hereby incorporated by reference in its entirety): 1 RLYCKNGGFFLRINPDGRVD GVREKSDPHI KLQLQAEERG VVSIKGVSAN RFLAMKEDGR 61 LLALKCATEECFFFERLESN NYNTYRSRKY SDWYVALKRT GQYKPGPKTG PGQKAILFLP 121 MSAKS Aminoacid sequence of Gallus gallus (chicken) FGF2 (SEQ ID NO: 152) (GenBankaccession no. NP_990764 1 maagaagsit tlpalpddgg ggafppghfk dpkrlycknggfflrinpdg rvdgvreksd 61 PHIKLQLQAE ERGVVSIKGV SANRFLAMKE DGRLLALKCATEECFFFERL ESNNYNTYRS 121 RKYSDWYVAL KRTGQYKPGP KTGPGQKAIL FLPMSAKSAmino acid sequence of Taeniopygia guttata (zebra finch) FGF2 (SEQ IDNO: 153) (GenBank accession no. XP_002188397, which is herebyincorporated by reference in its entirety): 1 MAAAGGIATL PDDGGSGAFPPGHFKDPKRL YCKNGGFFLR INPDGKVDGV REKSDPHIKL 61 QLQAEERGVV SIKGVSANRFLAMKEDGRLL ALKYATEECF FFERLESNNY NTYRSRKYSD 121 WYVALKRTGQ YKPGPKTGPGQKAILFLPMS AKS Amino acid sequence of Cynops pyrrhogaster (Japanesefirebelly newt) FGF2 (SEQ ID NO: 154) (GenBank accession no. BAB63249,which is hereby incorporated by reference in its entirety): 1 MAAGSITSLPALPEDGNGGT FTPGGFKEPK RLYCKNGGFF LRINSDGKVD GAREKSDSYI 61 KLQLQAEERGVVSIKGVCAN RYLAMKDDGR LMALKWITDE CFFFERLESN NYNTYRSRKY 121 SDWYVALKRTGQYKNGSKTG AGQKAILFLP MSAKS Amino acid sequence of Xenopus laevis(African clawed frog) FGF2 (SEQ ID NO: 155) (GenBank accession no.NP_001093341, which is hereby incorporated by reference in itsentirety): 1 MAAGSITTLP TESEDGGNTP FSPGSFKDPK RLYCKNGGFF LRINSDGRVDGSRDKSDSHI 61 KLQLQAVERG VVSIKGITAN RYLAMKEDGR LTSLRCITDE CFFFERLEANNYNTYRSRKY 121 SSWYVALKRT GQYKNGSSTG PGQKAILFLP MSAKS Amino acidsequence of Didelphis albiventris (white-eared opossum) FGF2 (SEQ ID NO:156) (GenBank accession no. ABL77404, which is hereby incorporated byreference in its entirety): 1 MAAGSITTLP ALSGDGGGGG AFPPGHFKDPKRLYCKNGGF FLRIHPDGRV DGIREKSDPN 61 IKLQLQAEER GVVSIKGVCA NRYLAMKEDGRLLALKYVTE ECFFFERLES NNYNTYRSRK 121 YSNWYVALKR TGQYKLGSKT GPGQKAILFSPCLLRC Amino acid sequence of Myotis lucifugus (microbat) FGF2 (partialamino acid sequence corresponding to human FGF2 residues 60 to 155) (SEQID NO: 157) (Ensembl accession no. ENSMLUP00000017859, which is herebyincorporated by reference in its entirety): 1 VKLQLQAEER GVVSIKGVCANRYLAMKEDG RLQASKCVTD ECFFFERLES NNYNTYRSRK 61 YSSWYVALKR NGQYKLGPKTGPGQKAILFL PMSAKS Amino acid sequence of Anolis carolinensis (anolelizard) FGF2 (partial amino acid sequence corresponding to human FGF2residues 16 to 155) (SEQ ID NO: 158) (Ensembl accession no.ENSACAP00000011657, which is heeby incorporated by reference in itsentirety): 1 rAAAASFPPGP FKDPKRLYCK NGGFFLRINP DGGVDGVREK SDPNIKLLLQAEERGVVSIK 61 GVCANRFLAM NEDGRLLALK YVTDECFFFE RLESNNYNTY RSRKYRDWYIALKRTGQYKL 121 GPKTGRGQKA ILFLPMSAKS Amino acid sequence of Dasypusnovemcinctus (armadillo) FGF2 (partial amino acid sequence correspondingto human FGF2 residues 1 to 94) (SEQ ID NO: 159) (Ensembl accession no.ENSDNOP00000011351, which is hereby incorporated by reference in itsentirety): 124    MAAGSIT TLPALPEDGG SGAFPPGHFK DPKRLYCKNG GFFLRIHPDGRVDGVREKSD 181 PNIKLQLQAE ERGVVSIKGV CANRYLAMRE DGRLQAS Amino acidsequence of Tupaia belangeri (tree shrew) FGF2 (SEQ ID NO: 160) (Ensemblaccession no. ENSTBEP00000000985, which is hereby incorporated byreference in its entirety): 1 AGVRAEREEA PGSGDSRGTD PAARSLIRRPDAAAREALLG ARSRVQGSST SWPASSRTGI 61 KLPDDSGQGM GGYPLDRPSR STGRGLGGAPDPAVKLQLQA EERGVVSIKG VCANRYLAMK 121 EDGRLLASKC VTDECFFFER LESNNYNTYRSRKYSSWYVA LKRTGQYKLG SKTGPGQKAI 181 LFLPMSAKS Amino acid sequence ofXenopus silurana tropicalis (western clawed frog) FGF2 (SEQ ID NO: 161)(GenBank accession no. NP_001017333, which is hereby incorporated byreference in its entirety): 1 MAAGSITTLP TESEDGNTPF PPGNFKDPKRLYCKNGGYFL RINSDGRVDG SRDKSDLHIK 61 LQLQAVERGV VSIKGITANR YLAMKEDGRLTSLKCITDEC FFYERLEANN YNTYRSRKNN 121 SWYVALKRTG QYKNGSTTGP GQKAILFLPMSAKS Amino acid sequence of Latimeria chalumnae (coelacanth) FGF2 (SEQID NO: 162) (Ensembl accession no. ENSLACP00000019200, which is herebyincorporated by reference in its entirety): 1 MAAGGITTLP AVPEDGGSSTFPPGNFKEPK RLYCKNGGYF LRINPDGRVD GTREKNDPYI 61 KLQLQAESIG VVSIKGVCSNRYLAMNEDCR LFGLKYPTDE CFFHERLESN NYNTYRSKKY 121 SDWYVALKRT GQYKPGPKTGLGQKAILFLP MSAKS Amino acid sequence of Tetraodon nigroviridis (spottedgreen pufferfish) FGF2 (SEQ ID NO: 163) (GenBank accession no. CAG04681,which is hereby incorporated by reference in its entirety): 34                                    MATGGIT TLPSTPEDGG SSGFPPGSFK 61DPKRLYCKNG GFFLRIKSDG VVDGIREKSD PHIKLQLQAT SVGEVVIKGV CANRYLAMNR 121DGRLFGTKRA TDECHFLERL ESNNYNTYRS RKYPTMFVGL TRTGQYKSGS KTGPGQKAIL 181FLPMSAKC Amino acid sequence of Gasterosteus aculeatus (stickleback)FGF2 (SEQ ID NO: 164) (Ensembl accession no. ENSGACP00000022078, whichis hereby incorporated by reference in its entirety): 1 MATAGFATLPSTPEDGGSGG FTPGGFKDPK RLYCKNGGFF LRIRSDGGVD GIREKSDAHI 61 KLQIQATSVGEVVIKGVCAN RYLAMNRDGR LFGVRRATDE CYFLERLESN NYNTYRSRKY 121 PGMYVALKRTGQYKSGSKTG PGQKAILFLP MSAKC Amino acid sequence of Takifugu rubripes(fugu rubripes) FGF2 (SEQ ID NO: 165) (GenBank accession no. CAD19830,which is hereby incorporated by reference in its entirety): 1 MATGGITTLPSTPEDGGSGG FPPGSFKDPK RLYCKNGGFF LRIRSDGAVD GTREKTDPHI 61 KLQLQATSVGEVVIKGVCAN RYLAMNRDGR LFGMKRATDE CHFLERLESN NYNTYRSRKY 121 PNMFVGLTRTGNYKSGTKTG PCQKAILFLP MSAKY Amino acid sequence of Oncorhynchus mykiss(rainbow trout) FGF2 (SEQ ID NO: 166) (GenBank accession no.NP_001118008, which is hereby incorporated by reference in itsentirety): 1 MATGEITTLP ATPEDGGSGG FLPGNFKEPK RLYCKNGGYF LRINSNGSVDGIRDKNDPHN 61 KLQLQATSVG EVVIKGVSAN RYLAMNADGR LFGPRRTTDE CYFMERLESNNYNTYRSRKY 121 PEMYVALKRT GQYKSGSKTG PGQKAILFLP MSARR Amino acidsequence of Salmo salar (salmon) FGF2 (SEQ ID NO: 167) (GenBankaccession no. ACJ02099, which is hereby incorporated by reference in itsentirety): 1 MATGEITTLP ATPEDGGSGG FPPGNFKDPK RLYCKNGGYF LRINSNGSVDGIREKNDPHK 61 QPQFVRAWTL QGVKRSTGML AHVDSNASHN CVKVAGCSLG EFGSMSNRPHNRRPRVATPA 121 QDLHIRLLHL RDRLKPATRT ADKTEEYFCL Amino acid sequence ofDanio rerio (zebrafish) FGF2 (SEQ ID NO: 168) (GenBank accession no.AAP32155, which is hereby incorporated by reference in its entirety): 1MATGGITTLP AAPDAENSSF PAGSFRDPKR LYCKNGGFFL RINADGRVDG ARDKSDPHIR 61LQLQATAVGE VLIKGICTNR FLAMNADGRL FGTKRTTDEC YFLERLESNN YNTYRSRKYP 121DWYVALKRTG QYKSGSKTSP GQKAILFLPM SAKC Amino acid sequence of Oreochromisniloticus (Nile tilapia) FGF2 (SEQ ID NO: 169) (GenBank accession no.XP_003443412, which is hereby incorporated by reference in itsentirety): 1 MATGGITTLP ATPEDGGSSG FPPGNFKDPK RLYCKNGGFF LRIKSDGGVDGIREKNDPHI 61 KLQLQATSVG EVVIKGICAN RYLAMNRDGR LFGARRATDE CYFLERLESNNYNTYRSRKY 121 PNMYVALKRT GQYKSGSKTG PGQKAILFLP MSAKC Amino acidsequence of Oryzias latipes (medaka) FGF2 (SEQ ID NO: 170) (Ensemblaccession no. ENSORLP00000025834, which is hereby incorporated byreference in its entirety): 1 MATGEITTLP SPAENSRSDG FPPGNYKDPKRLYCKNGGLF LRIKPDGGVD GIREKKDPHV 61 KLRLQATSAG EVVIKGVCSN RYLAMHGDGRLFGVRQATEE CYFLERLESN NYNTYRSKKY 121 PNMYVALKRT GQYKPGNKTG PGQKAILFLPMSAKY

As noted above, the portion of the paracrine FGF may be modified todecrease binding affinity for heparin and/or heparan sulfate compared tothe portion without the modification. In one embodiment, themodification of the paracrine FGF includes one or more substitutions,additions, or deletions.

In one embodiment, the modification is one or more substitutions locatedat one or more amino acid residues of SEQ ID NO: 121 selected from N36,K128, R129, K134, K138, Q143, K144, C78, C96, and combinations thereof.In one embodiment, the one or more substitutions are selected from N36T,K128D, R129Q, K134V, K138H, Q143M, K144T/L/I, C78S, C96S, andcombinations thereof. In one embodiment, the modification is one or moresubstitutions which are located at one or more amino acid residuescorresponding to residues of SEQ ID NO: 121 selected from N36, K128,R129, K134, K138, Q143, K144, C78, C96, and combinations thereof. In oneembodiment, the modification is one or more substitutions which arelocated at one or more amino acid residues corresponding to residues ofSEQ ID NO: 121 selected from N36, K128, R129, K134, K138, Q143, K144,C78, C96, and combinations thereof. Amino acid residues corresponding tothose of SEQ ID NO: 121 may be determined by, for example, sequenceanalysis and structural analysis.

It will be understood that the portion of the paracrine FGF according tothe present invention may be derived from a nucleotide sequence thatencodes a paracrine FGF protein. For example, in one embodiment,nucleotide sequence is the nucleotide sequence that encodes human FGF2(GenBank Accession No. NM_002006, which is hereby incorporated byreference in its entirety)(SEQ ID NO: 171), as follows:

468                                                    ATG GCAGCCGGGA481 GCATCACCAC GCTGCCCGCC TTGCCCGAGG ATGGCGGCAG CGGCGCCTTC CCGCCCGGCC541 ACTTCAAGGA CCCCAAGCGG CTGTACTGCA AAAACGGGGG CTTCTTCCTG CGCATCCACC601 CCGACGGCCG AGTTGACGGG GTCCGGGAGA AGAGCGACCC TCACATCAAG CTACAACTTC661 AAGCAGAAGA GAGAGGAGTT GTGTCTATCA AAGGAGTGTG TGCTAACCGT TACCTGGCTA721 TGAAGGAAGA TGGAAGATTA CTGGCTTCTA AATGTGTTAC GGATGAGTGT TTCTTTTTTG781 AACGATTGGA ATCTAATAAC TACAATACTT ACCGGTCAAG GAAATACACC AGTTGGTATG841 TGGCACTGAA ACGAACTGGG CAGTATAAAC TTGGATCCAA AACAGGACCT GGGCAGAAAG901 CTATACTTTT TCTTCCAATG TCTGCTAAGA GCTGA

In another embodiment of the present invention, the portion of theparacrine FGF of the chimeric protein may be derived from a nucleotidesequence that encodes an ortholog of human FGF2. Nucleotide sequencesthat encode FGF2 orthologs are shown in Table 4.

TABLE 4 Gorilla FGF2 gene coding sequence (amino acids (“aa”) 104-258)(SEQ ID NO: 172) (Ensembl accession no. ENSGGOT00000004842, which ishereby incorporated by reference in its entirety): 310            ATGGCAGCC GGGAGCATCA CCACGCTGCC CGCCTTGCCC GAGGATGGCG 359GCAGCGGCGC CTTCCCGCCC GGCCACTTCA AGGACCCCAA GCGGCTGTAC TGCAAAAACG 419GGGGCTTCTT CCTGCGCATC CACCCCGACG GCCGAGTTGA CGGGGTCCGG GAGAAGAGCG 479ACCCTCACAT CAAGCTACAA CTTCAAGCAG AAGAGAGAGG AGTTGTGTCT ATCAAAGGAG 539TGTGTGCTAA CCGTTACCTT GCTATGAAGG AAGATGGAAG ATTACTGGCT TCTAAATGTG 599TTACGGATGA GTGTTTCTTT TTTGAACGAT TGGAATCTAA TAACTACAAT ACTTACCGGT 659CAAGGAAATA CACCAGTTGG TATGTGGCAC TGAAACGAAC TGGGCAGTAT AAACTTGGAT 719CCAAAACAGG ACCTGGGCAG AAAGCTATAC TTTTTCTTCC AATGTCTGCT AAGAGCTGASumatran orangutan FGF2 gene coding sequence (aa 168-322) (SEQ ID NO:173) (GenBank accession no. XM_002815126, which is hereby incorporatedby reference in its entirety): 504                          ATGGCAGCCGGGAGCAT CACCACGCTG CCCGCCTTGC 541 CCGAGGATGG CGGCAGCGGC GCCTTCCCGCCGGGCCACTT CAAGGACCCC AAGCGGCTGT 601 ACTGCAAAAA CGGGGGCTTC TTCCTGCGCATCCACCCCGA CGGCCGAGTT GACGGGGTCC 661 GAGAGAAGAG CGACCCTCAC ATCAAACTACAACTTCAAGC AGAAGAAAGA GGAGTTGTGT 721 CTATCAAAGG AGTGTGTGCT AACCGCTACCTTGCTATGAA GGAAGATGGA AGATTACTGG 781 CTTCTAAATG TGTTACGGAT GAGTGTTTCTTTTTTGAACG ATTGGAATCT AATAACTACA 841 ATACTTACCG GTCAAGGAAA TACACCAGTTGGTATGTGGC ACTGAAACGA ACTGGGCAGT 901 ATAAACTTGG ATCCAAAACA GGACCTGGGCAGAAAGCTAT ACTTTTTCTT CCAATGTCTG 961 CTAAGAGCTG A Rhesus monkey FGF2gene coding sequence (aa 83-237) (SEQ ID NO: 174) (GenBank accession no.XM_001099284, which is hereby incorporated by reference in itsentirety): 247       ATGG CAGCCGGGAG CATCACCACG CTGCCCGCCT TGCCCGAGGATGGCGGCAGC 301 GGCGCCTTCC CGCCTGGCCA CTTCAAGGAC CCCAAGCGGC TGTACTGCAAAAACGGGGGC 361 TTCTTCCTGC GCATTCACCC CGACGGCCGA GTTGACGGGG TCCGGGAGAAGAGCGACCCT 421 CACATCAAAT TACAACTTCA AGCAGAAGAG AGAGGAGTTG TGTCTATCAAAGGAGTGTGT 481 GCTAACCGTT ACCTTGCTAT GAAGGAAGAT GGAAGATTAC TGGCTTCTAAATGTGTTACA 541 GATGAGTGTT TCTTTTTTGA ACGATTGGAA TCTAATAACT ACAATACTTACCGGTCAAGG 601 AAATACACCA GTTGGTATGT GGCACTGAAA CGAACTGGGC AATATAAACTTGGATCCAAA 661 ACAGGACCTG GGCAGAAAGC TATACTTTTT CTTCCAATGT CTGCTAAGAGCTGA Chimpanzee FGF2 gene coding sequence (aa 134-288) (SEQ ID NO: 175)(GenBank accession no. NM_001110241, which is hereby incorporated byreference in its entirety): 400                                          A TGGCAGCCGG GAGCATCACC 421ACGCTGCCCG CCTTGCCCGA GGATGGCGGC AGCGGCGCCT TCCCGCCCGG CCACTTCAAG 481GACCCCAAGC GGCTGTACTG CAAAAACGGG GGCTTCTTCC TGCGCATCCA CCCCGACGGC 541CGAGTTGACG GGGTCCGGGA GAAGAGCGAC CCTCACATCA AGCTACAACT TCAAGCAGAA 601GAGAGAGGAG TTGTGTCTAT CAAAGGAGTG TGTGCTAACC GTTACCTTGC TATGAAGGAA 661GATGGAAGAT TACTGGCTTC TAAATGTGTT ACGGATGAGT GTTTCTTTTT TGAACGATTG 721GAATCTAATA ACTACAATAC TTACCGGTCA AGGAAATACA CCAGTTGGTA TGTGGCACTG 781AAACGAACTG GGCAGTATAA ACTTGGATCC AAAACAGGAC CTGGGCAGAA AGCTATACTT 841TTTCTTCCAA TGTCTGCTAA GAGCTGA Pygmy chimpanzee FGF2 gene coding sequence(112-266) (SEQ ID NO: 176) (GenBank accession no. XM_003816433, which ishereby incorporated by reference in its entirety): 334                                    ATGGCAG CCGGGAGCAT CACCACGCTG 361CCCGCCTTGC CCGAGGATGG CGGCAGCGGC GCCTTCCCGC CCGGCCACTT CAAGGACCCC 421AAGCGGCTGT ACTGCAAAAA CGGGGGCTTC TTCCTGCGCA TCCACCCCGA CGGCCGAGTT 481GACGGGGTCC GGGAGAAGAG CGACCCTCAC ATCAAGCTAC AACTTCAAGC AGAAGAGAGA 541GGAGTTGTGT CTATCAAAGG AGTGTGTGCT AACCGTTACC TTGCTATGAA GGAAGATGGA 601AGATTACTGG CTTCTAAATG TGTTACGGAT GAGTGTTTCT TTTTTGAACG ATTGGAATCT 661AATAACTACA ATACTTACCG GTCAAGGAAA TACACCAGTT GGTATGTGGC ACTGAAACGA 721ACTGGGCAGT ATAAACTTGG ATCCAAAACA GGACCTGGGC AGAAAGCTAT ACTTTTTCTT 781CCAATGTCTG CTAAGAGCTG A Bolivian squirrel monkey FGF2 gene codingsequence (1-155) (SEQ ID NO: 177) (GenBank accession no. XM_003936241,which is hereby incorporated by reference in its entirety): 23                        ATGGCAGC CGGGAGCATC ACCACGCTGC CCGCCCTGCC 61CGAAGACGGC GGCAGCGGCG CCTTCCCGCC CGGCCACTTC AAAGACCCCA AGCGGCTGTA 121CTGCAAAAAC GGGGGCTTCT TCCTGCGAAT CCACCCCGAC GGCCGAGTGG ACGGGGTCCG 181GGAGAAGAGC GACCCTCACA TCAAACTACA ACTTCAAGCA GAAGAGAGAG GAGTTGTATC 241TATCAAAGGA GTGTGTGCTA ACCGTTACCT TGCTATGAAG GAAGATGGAA GATTACTGGC 301TTCTAAATGT GTTACGGACG AGTGTTTCTT TTTTGAACGA TTGGAATCTA ATAACTACAA 361TACTTACCGA TCAAGGAAAT ACACCAGTTG GTATGTGGCA CTGAAACGAA CTGGGCAGTA 421TAAACTTGGA TCCAAAACAG GACCTGGGCA GAAAGCTATA CTTTTTCTTC CAATGTCTGC 481TAAGAGCTGA Northern white-cheeked gibbon FGF2 gene coding sequence (aa1-155) (SEQ ID NO: 178) (GenBank accession no. XM_003271356, which ishereby incorporated by reference in its entirety): 435                                                   ATG GCAGCCGGGA 481GCATCACCAC GCTGCCCGCC TTGCCGGAGG ATGGCGGCAG CGGCGCCTTC CCGCCCGGCC 541ACTTCAAGGA CCCCAAGCGG CTGTACTGCA AAAACGGGGG TTTCTTCCTG CGCATCCACC 601CCGACGGTCG AGTTGACGGG GTCCGGGAGA AGAGCGACCC TCACATCAAA CTACAACTTC 661AAGCAGAAGA GAGAGGAGTT GTGTCTATCA AAGGAGTGTG TGCTAACCGT TACCTTGCTA 721TGAAGGAAGA TGGAAGATTA CTGGCTTCTA AATGTGTTAC GGATGAGTGT TTCTTTTTTG 781AACGATTGGA ATCTAATAAC TACAATACTT ACCGGTCAAG GAAATACACC AGTTGGTATG 841TGGCACTGAA ACGAACTGGG CAGTATAAAC TTGGATCCAA AACAGGACCT GGGCAGAAAG 901CTATACTTTT TCTTCCAATG TCTGCTAAGA GCTGA Horse FGF2 gene coding sequence(aa 1-155) (SEQ ID NO: 179) (GenBank accession no. NM_001195221, whichis hereby incorporated by reference in its entirety): 54                                                          ATGGCAG 61CCGGGAGCAT CACCACGCTG CCCGCCCTGC CCGAGGACGG CGGCAGCGGC GCCTTCCCGC 121CCGGCCACTT CAAGGACCCC AAGCGGCTCT ACTGCAAAAA CGGGGGCTTC TTCCTGCGCA 181TCCACCCCGA CGGCCGAGTG GACGGGGTCC GGGAGAAGAG CGACCCTCAC ATCAAACTAC 241AACTTCAAGC AGAAGAGAGA GGGGTTGTGT CTATCAAAGG AGTGTGTGCG AACCGTTATC 301TTGCTATGAA GGAAGATGGA AGGTTACTGG CTTCTAAATG TGTTACGGAC GAGTGTTTCT 361TTTTTGAACG ATTGGAATCT AATAACTACA ATACTTACCG GTCAAGGAAA TACTCCAGTT 421GGTATGTGGC CCTGAAACGA ACGGGGCAGT ATAAACTTGG ACCCAAAACA GGACCTGGAC 481AGAAAGCTAT ACTTTTTCTT CCAATGTCTG CTAAGAGCTG A Cattle FGF2 gene codingsequence (aa 1-155) (SEQ ID NO: 180) (GenBank accession no. NM_174056,which is hereby incorporated by reference in its entirety): 104                                               ATGGCCG CCGGGAGCAT 121CACCACGCTG CCAGCCCTGC CGGAGGACGG CGGCAGCGGC GCTTTCCCGC CGGGCCACTT 181CAAGGACCCC AAGCGGCTGT ACTGCAAGAA CGGGGGCTTC TTCCTGCGCA TCCACCCCGA 241CGGCCGAGTG GACGGGGTCC GCGAGAAGAG CGACCCACAC ATCAAACTAC AACTTCAAGC 301AGAAGAGAGA GGGGTTGTGT CTATCAAAGG AGTGTGTGCA AACCGTTACC TTGCTATGAA 361AGAAGATGGA AGATTACTAG CTTCTAAATG TGTTACAGAC GAGTGTTTCT TTTTTGAACG 421ATTGGAGTCT AATAACTACA ATACTTACCG GTCAAGGAAA TACTCCAGTT GGTATGTGGC 481ACTGAAACGA ACTGGGCAGT ATAAACTTGG ACCCAAAACA GGACCTGGGC AGAAAGCTAT 541ACTTTTTCTT CCAATGTCTG CTAAGAGCTG A Olive baboon FGF2 gene codingsequence (1-155) (SEQ ID NO: 181) (GenBank accession no. XM_003899161,which is hereby incorporated by reference in its entirety): 467                                                  ATGG CAGCCGGGAG 481CATCACCACG CTGCCCGCCT TGCCCGAGGA TGGCGGCAGC GGCGCCTTCC CGCCCGGCCA 541CTTCAAGGAC CCCAAGCGGC TGTACTGCAA AAACGGGGGC TTCTTCCTGC GCATTCACCC 601CGACGGCCGA GTTGACGGGG TCCGGGAGAA GAGCGACCCT CACATCAAAT TACAACTTCA 661AGCAGAAGAG AGAGGAGTTG TGTCTATCAA AGGAGTGTGT GCTAACCGTT ACCTTGCTAT 721GAAGGAAGAT GGAAGATTAC TGGCTTCTAA ATGTGTTACG GATGAGTGTT TCTTTTTTGA 781ACGATTGGAA TCTAATAACT ACAATACTTA CCGGTCAAGG AAATACACCA GTTGGTATGT 841GGCACTGAAA CGAACTGGGC AGTATAAACT TGGATCCAAA ACAGGACCTG GGCAGAAAGC 901TATACTTTTT CTTCCAATGT CTGCTAAGAG CTGA Alpaca FGF2 gene coding sequence(aa 111-265) (SEQ ID NO: 182) (Ensembl accession no. ENSVPAT00000010536,which is hereby incorporated by reference in its entirety): 341                                 ATGGCAGCTG GGAGCATCAC CACGCTGCCC 361GCCCTGCCGG AGGACGGCGG CAGCGGCGCC TTCCCGCCCG GCCACTTCAA GGACCCCAAG 421CGGTTGTACT GCAAAAACGG GGGCTTCTTC CTGCGCATCC ACCCCGACGG CCGAGTGGAC 481GGGGTCCGGG AGAAGAGCGA CCCTCACATC AAACTACAAC TTCAAGCAGA AGAGAGAGGG 541GTCGTGTCTA TCAAAGGAGT GTGTGCAAAC CGTTACCTTG CTATGAAGGA AGATGGAAGA 601TTACTGGCTT CTAAATGTGT CACAGACGAG TGTTTCTTTT TTGAACGATT GGAATCTAAT 661AACTACAATA CTTACCGGTC AAGGAAATAC TCCAGTTGGT ATGTGGCACT GAAACGAACT 721GGGCAGTACA AACTTGGACC CAAAACAGGA CCTGGGCAGA AAGCTATACT TTTCCTTCCA 781ATGTCTGCTA AGAGCTGA Sheep FGF2 gene coding sequence (aa 1-155) (SEQ IDNO: 183) (GenBank accession no. NM_001009769, which is herebyincorporated by reference in its entirety): 1 ATGGCCGCCG GGAGCATCACCACGCTGCCA GCCCTGCCGG AGGACGGCGG CAGCAGCGCT 61 TTCCCGCCCG GCCACTTTAAGGACCCCAAG CGGCTGTACT GCAAGAACGG GGGCTTCTTC 121 CTGCGCATCC ACCCCGACGGCCGAGTGGAC GGGGTCCGCG AGAAGAGCGA CCCTCACATC 181 AAACTACAAC TTCAAGCAGAAGAGAGAGGG GTTGTGTCTA TCAAAGGAGT GTGTGCAAAC 241 CGTTACCTTG CTATGAAAGAAGATGGAAGA TTACTAGCTT CTAAATGTGT TACAGACGAG 301 TGTTTCTTTT TTGAACGATTGGAGTCTAAT AACTACAATA CTTACCGGTC AAGGAAATAC 361 TCCAGTTGGT ATGTGGCACTGAAACGAACT GGGCAGTATA AACTTGGACC CAAAACAGGA 421 CCTGGGCAGA AAGCTATACTTTTTCTTCCA ATGTCTGCTA AGAGCTGA Western roe deer FGF2 gene codingsequence (1-108; partial amino acid sequence corresponding to human FGF2residues 42 to 149) (SEQ ID NO: 184) (GenBank accession no. AF152587,which is hereby incorporated by reference in its entirety): 1 GCGCATCCACCCCGACGGCC GAGTGGACGG GGTCCGCGAG AAGAGTGACC CTCACATCAA 61 ACTACAACTTCAAGCAGAAG AGAGAGGGGT TGTGTCTATC AAAGGAGTGT GTGCGAACCG 121 TTATCTTGCTATGAAAGAAG ACGGAAGATT ATTGGCTTCA AAATGTGTTA CAGACGAATG 181 TTTCTTTTTTGAACGATTGG AGTCTAATAA CTACAATACT TACCGGTCAA GGAAATACTC 241 CAGTTGGTATGTGGCACTGA AACGAACTGG GCAGTATAAA CTTGGACCCA AAACAGGACC 301 TGGGCAGAAAGCTATACTTT TTCTT Elephant FGF2 gene coding sequence (1-96; partial aminoacid sequence corresponding to human FGF2 residues 60 to 155) (SEQ IDNO: 185) (Ensembl accession no. ENSLAFT00000008249, which is herebyincorporated by reference in its entirety): 1 GTTAAACTAC AGCTTCAAGCAGAAGAGAGA GGTGTTGTGT CTATCAAAGG AGTGTGTGCC 61 AACCGTTATC TGGCTATGAAGGAAGATGGA AGATTGCTGG CTTCTAGATG TGTGACAGAT 121 GAATGTTTCT TCTTTGAACGACTGGAATCT AATAACTACA ATACTTACCG GTCAAGGAAA 181 TACACCAGTT GGTATGTGGCACTGAAACGA ACGGGGCAGT ATAAACTTGG ATCCAAAACA 241 GGACCTGGAC AGAAAGCTATACTTTTTCTT CCCATGTCTG CTAAGAGC Pig FGF2 gene coding sequence (1-120;partial amino acid sequence corresponding to human FGF2 residues 36 to155) (SEQ ID NO: 186) (GenBank accession no. AJ577089 and Ensemblaccession no. ENSSSCT00000009952, which is hereby incorporated byreference in its entirety): 1 GAACGGGGGC TTCTTCCTGC GCATCCACCCCGACGGCCGA GTGGATGGGG TCCGGGAGAA 61 GAGCGACCCT CACATCAAAC TACAACTTCAAGCAGAAGAG AGAGGGGTTG TGTCTATCAA 121 AGGAGTGTGT GCAAACCGTT ATCTTGCTATGAAGGAAGAT GGAAGATTAC TGGCTTCTAA 181 ATGTGTTACA GACGAGTGTT TCTTTTTTGAACGACTGGAA TCTAATAACT ACAATACTTA 241 CCGGTCGAGG AAATACTCCA GTTGGTATGTGGCACTGAAA CGAACTGGGC AGTATAAACT 301 TGGACCCAAA ACAGGACCTG GGCAGAAAGCTATACTTTTT CTTCCAATGT CTGCTAAGAG 361 C Panda FGF2 gene coding sequence(1-96; partial amino acid sequence corresponding to human FGF2 residues60 to 155) (SEQ ID NO: 187) (Ensembl accession no. ENSAMET00000019232,which is hereby incorporated by reference in its entirety): 1 GTCAAACTGCAACTTCAAGC GGAAGAGAGA GGGGTTGTAT CCATCAAAGG AGTATGTGCA 61 AATCGCTATCTTGCCATGAA GGAAGATGGA AGATTACTGG CTTCTAAATG TGTTACCGAT 121 GAGTGTTTCTTTTTTGAGCG ACTGGAATCT AATAACTACA ATACTTACCG GTCAAGGAAA 181 TACTCCAGTTGGTATGTGGC ACTGAAACGA ACTGGGCAGT ATAAACTTGG ACCCAAAACA 241 GGACCTGGGCAGAAAGCTAT ACTTTTTCTT CCAATGTCTG CTAAGAGC Sloth FGF2 gene codingsequence (aa 14-168) (SEQ ID NO: 188) (Ensembl accession no.ENSCHOT00000011394, which is hereby incorporated by reference in itsentirety): 40                                           A TGGCAGCCGGGAGCATCACC 61 ACGCTGCCCG CCCTGCCCGA GGACGGAGGC AGCGGCGCCT TACCGCCCGGCCACTTCAAA 121 GATCCCAAGC GGCTCTACTG CAAAAACGGG GGCTTCTTCC TGCGTATCCATCCCGACGGC 181 AGAGTGGACG GGGTCCGGGA GAAGAGCGAC CCCCACATCA AACTACAACTTCAAGCAGAA 241 GAGAGAGGGG TTGTGTCTAT CAAAGGTGTG TGTGCAAACC GATATCTTGCTATGAAGGAA 301 GATGGAAGAT TACAGGCTTC TAAATGTGTA ACGGACGAGT GTTTCTTTTTTGAACGATTG 361 GAATCTAATA ACTACAATAC GTACCGATCA AGGAAATACT CCAGTTGGTATGTGGCACTG 421 AAACGAACTG GGCAATATAA ACTTGGACCC AAAACAGGAC CTGGGCAGAAAGCCATACTT 481 TTTCTTCCAA TGTCTGCTAA GAGCTGA Water buffalo FGF2 genecoding sequence (aa 1-155) (SEQ ID NO: 189) (GenBank accession no.JQ326277, which is hereby incorporated by reference in its entirety): 1ATGGCCGCCG GGAGCATCAC CACGCTGCCA CCCCTGCCGG AGGACGGCGG CAGCGGCGCT 61TTCCCGCCCG GCCACTTCAA GGACCCCAAG CGGCTGTACT GCAAGAACGG GGGCTTCTTC 121CTGCGCATCC ACCCCGACGG CCGAGTGGAC GGGGTCCGCG AGAAGAGCGA CCCACACATC 181AAACTACAAC TTCAAGCAGA AGAGAGAGGG GTTGTGTCTA TCAAAGGAGT GTGTGCAAAC 241CGTTACCTTG CTATGAAAGA AGATGGAAGA TTACTAGCTT CCAAATGTGT TACAGACGAG 301TGTTTCTTTT TTGAACGATT GGAGTCTAGT AACTACAATA CTTACCGGTC AAGGAAATAC 361TCCAGTTGGT ATGTGGCACT GAAACGAACT GGGCAGTATA AACTTGGACC CAAAACAGGA 421CCTGGGCAGA AAGCTATACT TTTTCTTCCA ATGTCTGCTA AGAGCTGA Dog FGF2 genecoding sequence (aa 40-194) (SEQ ID NO: 190) (GenBank accession no.XM_003432481, which is hereby incorporated by reference in itsentirety): 118                                                              ATG 121GCAGCCGGGA GCATCACCAC GCTGCCCGCC CTGCCGGAGG ACGGCGGCAG CGGCGCCTTC 181CCGCCCGGCC ACTTCAAGGA CCCCAAGAGG CTGTACTGCA AAAAAGGGGG CTTCTTCCTG 241CGGATCCACC CCGACGGCCG GGTGGACGGG GTCCGGGAGA AGAGCGATCC CCACGTCAAA 301TTGCAACTTC AAGCAGAAGA GAGAGGCGTT GTGTCCATCA AAGGAGTATG TGCAAATCGC 361TATCTTGCTA TGAAGGAAGA TGGAAGATTA CTGGCTTCTA AATGTGTTAC TGACGAGTGC 421TTCTTTTTTG AACGATTGGA ATCTAATAAC TACAATACTT ACCGGTCAAG GAAATACTCC 481AGTTGGTATG TGGCACTGAA ACGAACTGGG CAGTATAAAC TTGGACCAAA AACAGGACCT 541GGGCAGAAAG CTATACTTTT TCTTCCAATG TCTGCTAAGA GCTGA Norway rat FGF2 genecoding sequence (aa 1-154) (SEQ ID NO: 191) (GenBank accession no.NM_019305, which is hereby incorporated by reference in its entirety):533                                                          ATGGCTGC541 CGGCAGCATC ACTTCGCTTC CCGCACTGCC GGAGGACGGC GGCGGCGCCT TCCCACCCGG601 CCACTTCAAG GATCCCAAGC GGCTCTACTG CAAGAACGGC GGCTTCTTCC TGCGCATCCA661 TCCAGACGGC CGCGTGGACG GCGTCCGGGA GAAGAGCGAC CCACACGTCA AACTACAGCT721 CCAAGCAGAA GAGAGAGGAG TTGTGTCCAT CAAGGGAGTG TGTGCGAACC GGTACCTGGC781 TATGAAGGAA GATGGACGGC TGCTGGCTTC TAAGTGTGTT ACAGAAGAGT GTTTCTTCTT841 TGAACGCCTG GAGTCCAATA ACTACAACAC TTACCGGTCA CGGAAATACT CCAGTTGGTA901 TGTGGCACTG AAACGAACTG GGCAGTATAA ACTCGGATCC AAAACGGGGC CTGGACAGAA961 GGCCATACTG TTTCTTCCAA TGTCTGCTAA GAGCTGA Naked mole-rat FGF2 genecoding sequence (1-134; partial amino acid sequence corresponding tohuman FGF2 residues 22 to 155) (SEQ ID NO: 192) (GenBank accession no.JH173674, which is hereby incorporated by reference in its entirety):378500                     C CACCCGGCCA CTTCAAGGAC CCAAAGCGGC 378531TGTACTGCAA AAACGGGGGC TTCTTCCTGC GCATCCACCC CGACGGCCGC 378581 GTGGACGGGGTCCGGGAGAA GAGCGACCCT CACG 418784    TCAAACT ACAACTTCAA GCAGAAGAGAGAGGAGTTGT GTCTATTAAG 418831 GGAGTGTGTG CGAACCGTTA CCTTGCTATG AAGGAAGATGGAAGATTACT 418881 GGCTTCT 433983   AAATGTGT TACAGATGAG TGTTTCTTTTTTGAACGATT GGAATCTAAT 434031 AACTACAATA CTTATCGGTC AAGGAAATAC TCCAGTTGGTATGTGGCACT 434081 GAAACGAACT GGACAATATA AACTTGGATC CAAAACAGGA CCGGGGCAGA434131 AAGCTATACT TTTTCTTCCA ATGTCTGCTA AGAGCTGA Bushbaby FGF2 genecoding sequence (aa 52-206) (SEQ ID NO: 193) (Ensembl accession no.ENSOGAT00000025228, which is hereby incorporated by reference in itsentirety): 154                                     ATGGCAG CCGGGAGCATCACCACGCTG 181 CCCTCCCTGC CCGAGGACGG CGGCAGCGAC GCCTTTCCGC CCGGCCACTTCAAGGACCCC 241 AAGCGACTGT ACTGCAAAAA CGGGGGCTTC TTCCTGCGCA TCCACCCCGACGGCCGAGTG 301 GACGGGGTCC GGGAGAAGAG CGACCCTTAC ATCAAACTAC AACTTCAAGCAGAAGAGAGA 361 GGAGTTGTGT CTATCAAAGG AGTGTGTGCG AACCGTTACC TTGCTATGAAGGAAGACGGA 421 AGATTGCTGG CTTCTAAATT GATTACAGAC GAGTGCTTCT TTTTTGAACGACTGGAATCT 481 AATAACTACA ATACTTACCG GTCAAGAAAA TACTCCAGTT GGTATGTGGCACTGAAACGA 541 ACTGGACAGT ATAAACTTGG ATCCAAAACA GGACCTGGGC AGAAAGCTATACTTTTTCTT 601 CCAATGTCTG CTAAGAGCTG A House mouse FGF2 gene codingsequence (aa 1-154) (SEQ ID NO: 194) (GenBank accession no. NM_008006,which is hereby incorporated by reference in its entirety): 198                  ATG GCTGCCAGCG GCATCACCTC GCTTCCCGCA CTGCCGGAGG 241ACGGCGGCGC CGCCTTCCCA CCAGGCCACT TCAAGGACCC CAAGCGGCTC TACTGCAAGA 301ACGGCGGCTT CTTCCTGCGC ATCCATCCCG ACGGCCGCGT GGATGGCGTC CGCGAGAAGA 361GCGACCCACA CGTCAAACTA CAACTCCAAG CAGAAGAGAG AGGAGTTGTG TCTATCAAGG 421GAGTGTGTGC CAACCGGTAC CTTGCTATGA AGGAAGATGG ACGGCTGCTG GCTTCTAAGT 481GTGTTACAGA AGAGTGTTTC TTCTTTGAAC GACTGGAATC TAATAACTAC AATACTTACC 541GGTCACGGAA ATACTCCAGT TGGTATGTGG CACTGAAACG AACTGGGCAG TATAAACTCG 601GATCCAAAAC GGGACCTGGA CAGAAGGCCA TACTGTTTCT TCCAATGTCT GCTAAGAGCT 661 GASquirrel FGF2 gene coding sequence (1-144; partial amino acid sequencecorresponding to human FGF2 residues 12 to 155) (SEQ ID NO: 195)(Ensembl accession no. ENSSTOT00000022105, which is hereby incorporatedby reference in its entirety): 1 CTGCCCGAGG ACGGCGGCGG CGGCGCCTTCCCGCCCGGCC ACTTTAAGGA CCCCAAGCGG 61 CTCTACTGCA AAAACGGAGG CTTCTTCCTGCGCATCCACC CCGACGGCCG AGTGGACGGG 121 GTCCGGGAGA AGAGCGACCC CCACATCAAGCTCCAGCTTC AAGCCGAAGA CCGAGGGGTT 181 GTGTCCATCA AGGGAGTGTG TGCAAACCGATACCTGGCCA TGAAGGAGGA CGGGAGGCTC 241 CTGGCTTCTA AATGTGTTAC GGACGAGTGTTTCTTTTTTG AACGACTGGA ATCAAATAAC 301 TACAATACTT ACCGGTCAAG GAAATACTCCAGTTGGTATG TGGCCCTGAA ACGAACAGGG 361 CAGTATAAAC TTGGATCCAA AACAGGACCTGGGCAGAAAG CTATACTTTT TCTTCCAATG 421 TCTGCTAAGA GC Domestic cat FGF2gene coding sequence (1-106; partial amino acid sequence correspondingto human FGF2 residues 25 to 130) (SEQ ID NO: 196) (GenBank accessionno. EU314952, which is hereby incorporated by reference in itsentirety): 1 CCACTTCAAG GACCCCAAGC GTCTGTACTG CAAAAACGGG GGCTTCTTCCTGCGCATCCA 61 CCCCGACGGC CGAGTGGATG GGGTCCGGGA GAAGAGCGAC CCTCACATCAAACTGCAACT 121 TCAGGCAGAA GAGAGAGGGG TTGTGTCCAT CAAAGGAGTC TGTGCAAACCGCTATCTTGC 181 CATGAAGGAA GATGGAAGAT TACTGGCTTC TAAATGTGTT ACGGACGAGTGTTTCTTTTT 241 TGAACGATTG GAATCTAATA ACTACAATAC TTATCGGTCA AGGAAATACTCCAGCTGGTA 301 TGTGGCACTG AAACGAAC Guinea pig FGF2 gene coding sequence(1-96; partial amino acid sequence corresponding to human FGF2 residues60 to 155) (SEQ ID NO: 197) (Ensembl accession no. ENSCPOT00000005443,which is hereby incorporated by reference in its entirety): 1 GTTAAACTACAACTTCAAGC CGAAGACAGA GGAGTTGTGT CTATCAAGGG AGTCTGTGCG 61 AACCGTTACCTTGCTATGAA GGAAGACGGA AGATTATTGG CTTCCAAATG TGTTACAGAT 121 GAATGTTTCTTTTTTGAACG ACTGGAATCT AATAACTACA ACACTTACCG GTCAAGGAAA 181 TACTCCAGTTGGTATGTGGC ACTGAAACGA ACTGGACAAT ATAAACTTGG GTCCAAAACA 241 GGACCAGGGCAGAAAGCCAT ACTTTTTCTT CCAATGTCTG CGAAGAGC Tasmanian devil FGF2 genecoding sequence (aa 48-203) (SEQ ID NO: 198) (Ensembl accession no.ENSSHAP00000012215, which is hereby incorporated by reference in itsentirety): 142                        ATGGCCGCG GGCAGCATCA CCACGTTGCCGGCCCTGGCC 181 GGGGATGGAG CCAGCGGGGG CGCCTTTCCC CCGGGCCACT TCCAGGACCCCAAGCGGCTG 241 TACTGCAAGA ACGGAGGCTT CTTCTTGCGC ATCCATCCCG ACGGTCACGTGGACGGCATC 301 CGCGAGAAGA GCGATCCGCA CATTAAACTT CAGCTTCAGG CAGAAGAGAGAGGAGTAGTG 361 TCTATTAAAG GAGTTTGTGC CAACCGCTAT CTTGCCATGA AAGAGGATGGCAGATTACTG 421 GCTCTGAAAT GTGTGACTGA AGAGTGTTTC TTCTTTGAAC GTCTAGAGTCCAACAATTAC 481 AACACTTATC GCTCAAGGAA ATACTCCAAT TGGTATGTGG CATTGAAACGCACAGGCCAG 541 TATAAGCTTG GATCCAAGAC TGGACCAGGG CAGAAAGCCA TCCTTTTCCTTCCCATGTCT 601 GCTAAGAGCT GA Gray short-tailed opossum FGF2 gene codingsequence (aa 1-155) (SEQ ID NO: 199) (GenBank accession no.NM_001033976, which is hereby incorporated by reference in itsentirety): 29                               AT GGCCGCAGGC AGCATCACCACGCTGCCAGC 61 CCTGTCCGGG GACGGAGGCG GCGGGGGCGC CTTTCCCCCG GGCCACTTCAAGGACCCCAA 121 GCGGCTGTAC TGCAAGAACG GAGGCTTCTT CCTGCGCATC CACCCCGACGGCCGTGTGGA 181 CGGCATCCGC GAGAAGAGCG ACCCGAACAT TAAACTACAA CTTCAGGCAGAAGAGAGAGG 241 AGTGGTGTCT ATTAAAGGAG TATGTGCCAA TCGCTATCTT GCCATGAAGGAAGATGGAAG 301 ATTATTGGCT TTGAAATATG TGACCGAAGA GTGTTTCTTT TTCGAACGCTTGGAGTCCAA 361 CAACTACAAC ACTTATCGCT CGAGGAAATA TTCCAATTGG TACGTGGCACTGAAACGAAC 421 GGGGCAGTAC AAGCTTGGAT CCAAGACTGG CCCGGGGCAG AAAGCCATCCTTTTCCTCCC 481 CATGTCTGCT AAGAGCTGA Rabbit FGF2 gene coding sequence (aa1-155) (SEQ ID NO: 200) (GenBank accession no. XM_002717238, which ishereby incorporated by reference in its entirety): 1 ATGGCAGCCGAGAGCATCAC CACGCTGCCC GCCCTGCCGG AGGATGGAGG CAGCGGCGCC 61 TTCCCGCCCGGCCACTTCAA GGACCCCAAG CGGCTGTACT GCAAAAACGG GGGTTTCTTC 121 CTGCGTATCCACCCCGACGG CCGCGTGGAC GGGGTCCGGG AGAAGAGCGA CCCACACATC 181 AAATTACAACTTCAAGCAGA AGAGAGAGGA GTTGTATCCA TCAAAGGTGT GTGTGCAAAC 241 CGTTACCTTGCTATGAAGGA AGATGGAAGA CTGCTGGCTT CTAAATGTGT TACAGACGAG 301 TGCTTCTTTTTTGAACGACT GGAGTCTAAT AACTACAATA CTTACCGGTC AAGGAAATAT 361 TCCAGCTGGTATGTGGCACT GAAACGAACT GGGCAGTATA AACTTGGATC CAAAACAGGA 421 CCTGGGCAGAAGGCTATACT TTTTCTTCCA ATGTCTGCTA AGAGCTGA Turkey FGF2 gene codingsequence (1-125; partial amino acid sequence corresponding to human FGF2residues 31 to 155) (SEQ ID NO: 201) (Ensembl accession no.ENSMGAT00000011845, which is hereby incorporated by reference in itsentirety): 1 CGGCTCTACT GTAAGAACGG CGGCTTCTTC CTGCGCATCA ATCCCGACGGCAGAGTGGAC 61 GGCGTCCGCG AGAAGAGCGA TCCGCACATC AAACTGCAGC TTCAGGCAGAAGAAAGAGGA 121 GTGGTATCAA TCAAAGGTGT AAGTGCAAAC CGCTTTCTGG CTATGAAGGAGGATGGCAGA 181 TTGCTGGCAC TGAAATGTGC AACAGAAGAA TGTTTCTTTT TTGAGCGTTTGGAATCTAAT 241 AATTATAACA CTTACCGGTC ACGGAAGTAC TCTGATTGGT ATGTGGCACTGAAAAGAACT 301 GGACAGTACA AGCCCGGACC AAAAACTGGA CCTGGACAGA AAGCTATCCTTTTTCTTCCA 361 ATGTCTGCTA AAAGC Gallus gallus FGF2 gene coding sequence(aa 1-158) (SEQ ID NO: 202) (GenBank accession no. NM_205433, which ishereby incorporated by reference in its entirety): 98                                        ATG GCGGCGGGGG CGGCGGGGAG 121CATCACCACG CTGCCGGCGC TGCCCGACGA CGGGGGCGGC GGCGCTTTTC CCCCCGGGCA 181CTTCAAGGAC CCCAAGCGGC TCTACTGCAA GAACGGCGGC TTCTTCCTGC GCATCAACCC 241CGACGGCAGG GTGGACGGCG TCCGCGAGAA GAGCGATCCG CACATCAAAC TGCAGCTTCA 301AGCAGAAGAA AGAGGAGTAG TATCAATCAA AGGCGTAAGT GCAAACCGCT TTCTGGCTAT 361GAAGGAGGAT GGCAGATTGC TGGCACTGAA ATGTGCAACA GAGGAATGTT TCTTTTTCGA 421GCGCTTGGAA TCTAATAACT ATAACACTTA CCGGTCACGG AAGTACTCTG ATTGGTATGT 481GGCACTGAAA AGGACTGGAC AGTACAAGCC CGGACCAAAA ACTGGACCTG GACAGAAAGC 541TATCCTTTTT CTTCCAATGT CTGCTAAAAG CTGA Zebra finch FGF2 gene codingsequence (aa 1-153) (SEQ ID NO: 203) (GenBank accession no.XM_002188361, which is hereby incorporated by reference in itsentirety): 1 ATGGCGGCGG CGGGGGGCAT CGCTACGCTG CCCGACGACG GCGGCAGCGGCGCCTTTCCC 61 CCGGGGCACT TCAAGGACCC CAAGCGCCTG TACTGCAAGA ACGGCGGCTTCTTCCTGCGC 121 ATCAACCCCG ACGGGAAGGT GGACGGCGTC CGCGAGAAGA GCGACCCGCACATCAAGCTG 181 CAGCTTCAGG CGGAGGAACG AGGAGTGGTG TCCATCAAAG GTGTCAGTGCCAATCGCTTC 241 CTGGCCATGA AAGAGGATGG CAGATTGCTG GCCTTGAAAT ATGCAACAGAAGAATGTTTC 301 TTTTTTGAAC GTTTGGAATC CAATAACTAT AACACTTACC GGTCACGGAAATACTCGGAT 361 TGGTATGTGG CACTGAAAAG AACTGGACAG TACAAACCTG GACCAAAAACTGGACCTGGA 421 CAGAAAGCTA TCCTTTTCCT TCCTATGTCT GCTAAAAGCT GA Japanesefirebelly newt FGF2 gene coding sequence (aa 1-155) (SEQ ID NO: 204)(GenBank accession no. AB064664, which is hereby incorporated byreference in its entirety): 384                          ATGGCTGCTGGGAGCAT CACCAGTCTC CCTGCCCTAC 421 CCGAGGACGG GAATGGCGGC ACCTTCACACCCGGCGGATT CAAAGAGCCG AAGAGGCTGT 481 ACTGCAAGAA CGGGGGCTTC TTTCTCCGGATCAACTCCGA CGGCAAGGTG GACGGAGCCC 541 GGGAGAAGAG CGACTCCTAC ATTAAACTGCAGCTTCAAGC AGAAGAGCGC GGTGTGGTGT 601 CCATCAAGGG AGTATGTGCA AACCGCTATCTCGCTATGAA GGATGATGGC AGGCTGATGG 661 CGCTGAAATG GATAACCGAT GAATGCTTCTTTTTCGAGCG ACTGGAGTCC AACAACTATA 721 ACACGTATCG ATCACGGAAA TATTCCGATTGGTATGTGGC GCTGAAAAGA ACTGGGCAAT 781 ACAAAAATGG ATCAAAAACC GGAGCAGGACAGAAAGCAAT CCTTTTTCTA CCCATGTCGG 841 CCAAGAGTTG A African clawed frogFGF2 gene coding sequence (aa 1-155) (SEQ ID NO: 205) (GenBank accessionno. NM_001099871, which is hereby incorporated by reference in itsentirety): 335                                      ATGGCG GCAGGGAGCATCACAACTCT 361 GCCAACTGAA TCCGAGGATG GGGGAAACAC TCCTTTTTCA CCAGGGAGTTTTAAAGACCC 421 CAAGAGGCTC TACTGCAAGA ACGGGGGCTT CTTCCTCAGG ATAAACTCAGACGGGAGAGT 481 GGACGGGTCA AGGGACAAAA GTGACTCGCA CATAAAATTA CAGCTACAAGCTGTAGAGCG 541 GGGAGTGGTA TCAATAAAGG GAATCACTGC AAATCGCTAC CTTGCCATGAAGGAAGATGG 601 GAGATTAACA TCGCTGAGGT GTATAACAGA TGAATGCTTC TTTTTTGAACGACTGGAAGC 661 TAATAACTAC AACACTTACC GGTCTCGGAA ATACAGCAGC TGGTATGTGGCACTAAAGCG 721 AACCGGGCAG TACAAAAATG GATCGAGCAC TGGACCGGGA CAAAAAGCTATTTTATTTCT 781 CCCAATGTCC GCAAAGAGCT GA White-eared opossum FGF2 genecoding sequence (aa 1-156) (SEQ ID NO: 206) (GenBank accession no.EF057322, which is hereby incorporated by reference in its entirety): 1ATGGCAGCAG GCAGCATCAC CACATTGCCG GCCCTGTCCG GGGACGGAGG CGGCGGGGGA 61GCCTTTCCTC CAGGCCACTT CAAGGACCCC AAGCGGCTGT ACTGCAAGAA CGGAGGCTTC 121TTCCTGCGCA TCCACCCCGA CGGCCGCGTG GACGGCATCC GCGAGAAGAG CGACCCGAAC 181ATTAAACTAC AACTTCAGGC AGAAGAGAGA GGAGTAGTGT CTATTAAAGG AGTATGTGCC 241AACCGATATC TTGCCATGAA GGAGGATGGC AGATTATTGG CTTTGAAATA TGTGACCGAA 301GAGTGTTTCT TTTTTGAACG TTTGGAGTCC AACAACTACA ACACTTATCG CTCAAGAAAA 361TATTCCAATT GGTATGTGGC ACTGAAACGA ACGGGGCAGT ATAAGCTTGG ATCCAAGACT 421GGCCCGGGGC AGAAAGCCAT CCTTTTCTCC CCATGTCTGC TAAGATGCTG A Microbat FGF2gene coding sequence (1-96; partial amino acid sequence corresponding tohuman FGF2 residues 60 to 155) (SEQ ID NO: 207) (Ensembl accession no.ENSMLUT00000027717, which is hereby incorporated by reference in itsentirety): 1 GTCAAACTCC AACTTCAAGC AGAAGAGAGA GGGGTCGTGT CTATCAAAGGAGTGTGTGCC 61 AACCGCTATC TCGCTATGAA GGAGGACGGC CGGTTACAGG CTTCTAAATGTGTTACGGAT 121 GAGTGTTTCT TTTTTGAACG GTTGGAATCC AATAACTACA ACACTTACCGGTCAAGAAAG 181 TACTCCAGTT GGTATGTGGC ATTGAAGCGG AATGGGCAGT ATAAACTTGGACCCAAAACA 241 GGACCTGGCC AGAAAGCCAT ACTTTTTCTT CCCATGTCTG CTAAGAGCAnole lizard FGF2 gene coding sequence (1-140; partial amino acidsequence corresponding to human FGF2 residues 16 to 155) (SEQ ID NO:208) (Ensembl accession no. ENSACAT00000011897, which is herebyincorporated by reference in its entirety): 1 GCGGCGGCGG CCTCTTTCCCCCCGGGCCCC TTCAAGGACC CCAAGCGCCT CTACTGCAAG 61 AACGGGGGCT TCTTCCTGCGGATCAACCCC GACGGCGGCG TGGACGGCGT CCGAGAGAAG 121 AGCGACCCCA ACATCAAATTGCTGCTCCAG GCAGAGGAGA GAGGTGTAGT GTCCATCAAA 181 GGTGTATGCG CAAACCGTTTCCTGGCTATG AATGAAGACG GTCGATTGTT AGCACTGAAA 241 TACGTAACAG ATGAATGCTTCTTTTTTGAA CGCTTGGAAT CTAATAATTA CAATACTTAT 301 CGGTCTCGTA AATACCGTGATTGGTACATT GCACTGAAAC GAACTGGTCA GTACAAACTT 361 GGACCAAAAA CTGGACGAGGCCAGAAAGCT ATCCTTTTCC TTCCAATGTC TGCCAAAAGT Armadillo FGF2 gene codingsequence (124-217; partial amino acid sequence corresponding to humanFGF2 residues 1 to 94) (SEQ ID NO: 209) (Ensembl accession no.ENSDNOT00000014647, which is hereby incorporated by reference in itsentirety): 361          A TGGCAGCCGG GAGCATCACC ACGCTGCCCG CTCTGCCCGAGGACGGCGGC 421 AGCGGCGCCT TCCCGCCGGG CCACTTCAAG GACCCCAAGC GGCTGTACTGCAAAAACGGG 481 GGCTTCTTCC TGCGCATCCA TCCCGACGGC CGAGTGGACG GGGTCCGGGAGAAGAGCGAC 541 CCTAACATCA AACTACAACT TCAAGCAGAA GAGAGAGGGG TCGTGTCTATCAAAGGCGTG 601 TGTGCGAACC GTTACCTTGC TATGCGGGAA GACGGAAGAC TCCAGGCGTC TTree shrew FGF2 gene coding sequence (1-189) (SEQ ID NO: 210) (Ensemblaccession no. ENSTBET00000001143, which is hereby incorporated byreference in its entirety): 1 GCGGGGGTTA GAGCTGAGAG GGAGGAGGCACCGGGGAGCG GTGACAGCCG GGGGACCGAT 61 CCCGCCGCGC GTTCGCTCAT CAGGAGGCCGGATGCTGCAG CGCGAGAGGC GCTTCTTGGA 121 GCCAGGAGCC GGGTTCAGGG CAGCTCCACCTCCTGGCCAG CCTCGTCACG AACCGGGATC 181 AAGTTGCCGG ACGACTCAGG TCAAGGAATGGGCGGCTATC CTCTGGACCG CCCGAGCCGG 241 AGCACAGGGC GAGGGCTGGG CGGTGCCCCGGACCCTGCCG TAAAACTACA GCTTCAAGCG 301 GAAGAGAGAG GGGTCGTGTC TATCAAAGGAGTGTGTGCAA ACCGTTACCT GGCCATGAAG 361 GAGGATGGGC GACTGCTGGC TTCTAAATGTGTTACAGATG AGTGTTTCTT TTTTGAACGA 421 CTGGAATCTA ATAACTACAA TACTTACCGGTCCCGAAAGT ACTCCAGCTG GTATGTGGCA 481 CTGAAACGAA CTGGGCAGTA TAAACTTGGATCCAAAACAG GACCTGGGCA GAAAGCTATA 541 CTTTTTCTTC CAATGTCTGC TAAAAGCWestern clawed frog FGF2 gene coding sequence (aa 1-154) (SEQ ID NO:211) (GenBank accession no. NM_001017333, which is hereby incorporatedby reference in its entirety): 171                                                       ATGGCAGCAG 181GAAGCATCAC AACCCTACCA ACCGAATCTG AGGATGGAAA CACTCCTTTC CCACCGGGGA 241ACTTTAAGGA CCCCAAGAGG CTCTACTGCA AGAATGGGGG CTACTTCCTC AGGATTAACT 301CAGACGGGAG AGTGGACGGA TCAAGGGATA AAAGTGACTT ACACATAAAA TTACAGCTAC 361AAGCAGTAGA GCGGGGAGTG GTATCAATAA AGGGAATCAC TGCAAATCGC TACCTTGCCA 421TGAAGGAAGA TGGGAGATTA ACATCGCTGA AGTGTATAAC AGATGAATGC TTCTTTTATG 481AACGATTGGA AGCTAATAAC TACAACACTT ACCGGTCTCG GAAAAACAAC AGCTGGTATG 541TGGCACTAAA GCGAACTGGG CAGTATAAAA ATGGATCGAC CACTGGACCA GGACAAAAAG 601CTATTTTGTT TCTCCCAATG TCAGCAAAAA GCTGA Coelacanth FGF2 gene codingsequence (aa 1-155) (SEQ ID NO: 212) (Ensembl accession no.ENSLACT00000019333, which is hereby incorporated by reference in itsentirety): 1                       ATGGCTGCGG GAGGAATCAC TACCCTGCCGGCGGTACCTG 41 AGGATGGAGG CAGCAGCACC TTCCCTCCAG GAAACTTCAA GGAGCCCAAGAGACTTTACT 101 GTAAGAATGG AGGCTATTTC TTAAGGATAA ACCCCGATGG AAGAGTGGATGGAACAAGGG 161 AGAAAAATGA TCCTTATATA AAATTACAAC TGCAAGCTGA ATCTATAGGAGTGGTGTCGA 221 TAAAGGGAGT TTGTTCAAAC CGTTACCTAG CGATGAATGA AGACTGTAGACTTTTTGGAT 281 TGAAATATCC AACGGATGAA TGTTTCTTCC ATGAGAGGCT GGAGTCCAACAACTACAATA 341 CTTATCGTTC AAAGAAGTAT TCGGATTGGT ATGTGGCGCT GAAACGGACTGGTCAGTACA 401 AACCTGGGCC AAAAACTGGA CTGGGACAAA AAGCAATCCT TTTCCTTCCGATGTCTGCCA 461 AGAGTTGA Spotted green pufferfish FGF2 gene codingsequence (aa 34-188) (SEQ ID NO: 213) (Ensembl accession no.ENSTNIT00000016254, which is hereby incorporated by reference in itsentirety): 1 ATGGCCACGG GAGGGATCAC GACGCTTCCA TCCACACCTG AAGACGGCGGCAGCAGCGGC 61 TTTCCTCCCG GCAGCTTCAA GGATCCCAAA AGGCTCTACT GTAAAAACGGAGGTTTCTTC 121 CTGAGGATCA AGTCCGACGG GGTCGTGGAC GGAATCCGGG AGAAGAGTGACCCCCACATA 181 AAGCTTCAGC TCCAGGCGAC CTCTGTGGGG GAGGTGGTCA TCAAGGGGGTGTGCGCTAAC 241 CGCTATCTGG CCATGAACAG AGATGGACGG CTGTTCGGAA CGAAACGAGCCACGGACGAA 301 TGCCATTTCT TAGAGCGGCT TGAGAGCAAC AACTACAACA CTTACCGCTCCAGGAAGTAC 361 CCAACCATGT TTGTGGGACT GACGCGGACG GGCCAGTACA AGTCTGGGAGCAAAACTGGA 421 CCGGGCCAAA AGGCCATCCT TTTTCTTCCG ATGTCCGCCA AATGCTAAStickleback FGF2 gene coding sequence (aa 1-155) (SEQ ID NO: 214)(Ensembl accession no. ENSGACT00000022120, which is hereby incorporatedby reference in its entirety): 1                    AT GGCCACGGCAGGCTTCGCGA CGCTTCCCTC CACGCCCGAA 43 GACGGCGGCA GCGGCGGCTT CACCCCCGGGGGATTCAAGG ATCCCAAGAG GCTGTACTGC 103 AAAAACGGGG GCTTCTTCTT GAGGATCAGGTCCGACGGAG GTGTAGATGG AATCAGGGAG 163 AAGAGCGACG CCCACATAAA GCTCCAAATCCAGGCGACGT CGGTGGGGGA GGTGGTCATC 223 AAAGGAGTCT GTGCCAACCG CTATCTGGCCATGAACAGAG ACGGCCGGCT GTTCGGAGTG 283 AGACGGGCGA CGGACGAATG CTACTTCCTGGAGCGGCTGG AGAGTAACAA CTACAACACC 343 TACCGCTCCA GGAAGTACCC CGGCATGTACGTGGCTCTGA AGCGGACCGG CCAGTACAAG 403 TCCGGGAGCA AAACCGGACC CGGTCAAAAGGCCATTCTGT TCCTCCCCAT GTCGGCTAAG 463 TGCTAA Fugu rubripes FGF2 genecoding sequence (aa 1-155) (SEQ ID NO: 215) (Ensembl accession no.ENSTRUT00000022363, which is hereby incorporated by reference in itsentirety): 127       ATGG CCACGGGAGG GATCACAACA CTTCCATCCA CACCTGAAGACGGCGGCAGC 181 GGCGGTTTTC CTCCCGGGAG CTTCAAGGAT CCCAAAAGGC TGTACTGTAAAAACGGCGGC 241 TTCTTCCTGA GGATCAGGTC CGACGGGGCC GTGGACGGAA CCCGGGAGAAGACTGACCCC 301 CACATAAAGC TTCAGCTCCA GGCGACCTCT GTGGGGGAGG TGGTCATCAAGGGGGTTTGT 361 GCTAATCGTT ATCTGGCCAT GAACAGAGAT GGACGACTGT TTGGAATGAAACGAGCGACG 421 GATGAATGCC ACTTCTTAGA GCGGCTCGAG AGCAACAACT ACAACACCTACCGCTCCAGG 481 AAGTACCCCA ACATGTTTGT GGGACTGACG CGAACTGGCA ACTACAAGTCTGGGACTAAA 541 ACTGGACCGG GCCAAAAGGC CATCCTCTTT CTTCCGATGT CGGCCAAATACTAA Rainbow trout FGF2 gene coding sequence (aa 1-155) (SEQ ID NO: 216)(GenBank accession no. NM_001124536, which is hereby incorporated byreference in its entirety): 390                                ATGGCCACAGG AGAAATCACC ACTCTACCCG 421 CCACACCTGA AGATGGAGGC AGTGGCGGCTTCCTTCCAGG AAACTTTAAG GAGCCCAAGA 481 GGTTGTACTG TAAAAATGGA GGCTACTTCTTGAGGATAAA CTCTAACGGA AGCGTGGACG 541 GGATCAGAGA TAAGAACGAC CCCCACAATAAGCTTCAACT CCAGGCGACC TCAGTGGGGG 601 AAGTAGTAAT CAAAGGGGTC TCAGCCAACCGCTATCTGGC CATGAATGCA GATGGAAGAC 661 TGTTTGGACC GAGACGGACA ACAGATGAATGCTACTTCAT GGAGAGGCTG GAGAGTAACA 721 ACTACAACAC CTACCGCTCT CGAAAGTACCCTGAAATGTA TGTGGCACTG AAAAGGACTG 781 GCCAGTACAA GTCAGGATCC AAAACTGGACCCGGCCAAAA AGCCATCCTC TTCCTCCCCA 841 TGTCAGCCAG ACGCTGA Salmon FGF2 genecoding sequence (1-150) (SEQ ID NO: 217) (GenBank accession no.EU816603, which is hereby incorporated by reference in its entirety):99402                                              ATGGCCACA GGAGAAATCA99421 CCACTCTACC CGCCACACCT GAAGATGGAG GCAGTGGCGG CTTCCCTCCA GGAAACTTTA99481 AGGATCCCAA GAGGCTGTAC TGTAAAAACG GGGGCTACTT CTTGAGAATA AACTCTAATG99541 GAAGCGTGGA CGGGATCCGA GAGAAGAACG ACCCCCACA 100968                                                   AAC AGCCTCAATT 100981TGTCAGGGCA TGGACTCTTC AAGGTGTCAA ACGTTCCACA GGGATGCTGG CCCATGTTGA 101041CTCCAACGCT TCCCACAATT GTGTCAAGGT GGCTGGATGT TCTTTGGGAG 101845                          AATTTG GCAGTATGTC CAACCGGCCT CATAACCGCA 101881GACCACGTGT AGCTACACCA GCCCAGGACC TCCACATCCG GCTTCTTCAT CTACGGGATC 101941GTCTGAAACC AGCCACCCGA ACAGCTGATA AAACTGAGGA GTATTTCTGT CTGTAA ZebrafishFGF2 gene coding sequence (aa 1-154) (SEQ ID NO: 218) (GenBank accessionno. AY269790, which is hereby incorporated by reference in itsentirety): 43                                               ATGGCCACCGGAGGGATC 61 ACCACACTCC CGGCCGCTCC GGACGCCGAA AACAGCAGCT TTCCCGCGGGCAGCTTCAGG 121 GATCCCAAGC GCCTGTACTG CAAAAACGGA GGATTCTTCC TGCGGATCAACGCGGACGGC 181 CGAGTGGACG GAGCCCGAGA CAAGAGCGAC CCGCACATTC GTCTGCAGCTGCAGGCGACG 241 GCAGTGGGTG AAGTACTCAT TAAAGGCATC TGTACCAACC GTTTCCTTGCCATGAACGCA 301 GACGGACGAC TGTTCGGGAC GAAAAGGACC ACAGATGAAT GTTATTTCCTGGAGCGCCTG 361 GAGTCCAACA ACTACAACAC ATACAGATCC CGCAAGTATC CCGACTGGTACGTGGCTCTG 421 AAGAGAACCG GCCAGTATAA AAGCGGCTCT AAAACCAGCC CGGGACAGAAGGCCATCCTG 481 TTTCTGCCCA TGTCGGCCAA ATGCTGA Nile tilapia FGF2 genecoding sequence (aa 1-155) (SEQ ID NO: 219) (GenBank accession no.XM_003443364, which is hereby incorporated by reference in itsentirety): 1 ATGGCCACGG GAGGAATCAC AACACTTCCC GCTACACCTG AAGACGGCGGCAGCAGCGGC 61 TTTCCTCCTG GGAACTTCAA GGACCCTAAA AGGCTGTACT GTAAAAATGGTGGCTTCTTC 121 TTGAGGATAA AATCTGATGG AGGAGTGGAT GGAATACGAG AGAAAAACGACCCCCACATA 181 AAGCTTCAAC TCCAGGCGAC CTCAGTGGGA GAAGTGGTCA TCAAAGGGATTTGTGCAAAC 241 CGATATCTGG CAATGAACAG AGATGGACGA CTGTTTGGAG CGAGAAGAGCAACAGATGAG 301 TGCTACTTCT TAGAGCGGCT CGAGAGCAAC AACTACAACA CCTACCGCTCCAGGAAGTAC 361 CCAAACATGT ACGTGGCGCT GAAGCGGACT GGCCAGTACA AGTCTGGAAGCAAAACTGGA 421 CCGGGTCAAA AGGCAATTCT CTTTCTCCCA ATGTCTGCTA AATGCTAAMedaka FGF2 gene coding sequence (aa 1-155) (SEQ ID NO: 220) (Ensemblaccession no. ENSORLT00000025835, which is hereby incorporated byreference in its entirety): 1 ATGGCTACGG GAGAAATCAC AACACTTCCCTCCCCAGCTG AAAACAGCAG AAGCGATGGC 61 TTTCCTCCAG GGAACTACAA GGATCCTAAGAGGCTCTACT GTAAAAATGG AGGTTTGTTT 121 TTGAGGATTA AACCTGATGG AGGAGTGGATGGAATCCGGG AAAAAAAAGA TCCCCACGTT 181 AAGCTTCGCC TTCAGGCTAC CTCAGCGGGAGAGGTGGTGA TCAAAGGAGT TTGTTCAAAC 241 AGATATCTGG CGATGCATGG AGATGGACGTCTATTTGGAG TGAGACAAGC AACAGAGGAA 301 TGCTACTTCT TGGAGCGACT AGAGAGCAACAACTATAACA CCTATCGCTC TAAAAAGTAC 361 CCAAACATGT ACGTGGCACT GAAGCGGACAGGCCAGTACA AACCTGGAAA CAAAACTGGA 421 CCAGGTCAAA AGGCCATTCT CTTTCTGCCTATGTCTGCCA AGTACTAA

As noted above, also encompassed within the present invention areportions of paracrine FGFs other than FGF1 and/or FGF2 (e.g., FGF4,FGF5, FGF6, FGF9, FGF16, and FGF20). The portion of the paracrine FGFmay be from human FGF4, FGF5, FGF6, FGF9, FGF16, and/or FGF20 having theamino acid sequences shown in Table 5, or orthologs thereof.

TABLE 5 Amino acid sequence of human FGF4(SEQ ID NO: 221) (GenBank accession no. NP 001998, which is hereby incorporated by reference in its entirety):   1 MSGPGTAAVA LLPAVLLALL APWAGRGGAA APTAPNGTLE AELERRWESL VALSLARLPV  61 AAQPKEAAVQ SGAGDYLLGI KRLRRLYCNV GIGFHLQALP DGRIGGAHAD TRDSLLELSP 121 VERGVVSIFG VASRFFVAMS SKGKLYGSPF FTDECTFKEI LLPNNYNAYE SYKYPGMFIA 181 LSKNGKTKKG NRVSPTMKVT HFLPRL Amino acid sequence of human FGF5(SEQ ID NO: 222) (GenBank Accession No. NP 004455, which is hereby incorporated by reference in its entirety):   1 MSLSFLLLLF FSHLILSAWA HGEKRLAPKG QPGPAATDRN PRGSSSRQSS SSAMSSSSAS  61 SSPAASLGSQ GSGLEQSSFQ WSPSGRRTGS LYCRVGIGFH LQIYPDGKVN GSHEANMLSV 121 LEIFAVSQGI VGIRGVFSNK FLAMSKKGKL HASAKFTDDC KFRERFQENS YNTYASAIHR 181 TEKTGREWYV ALNKRGKAKR GCSPRVKPQH ISTHFLPRFK QSEQPELSFT VTVPEKKKPP 241 SPIKPKIPLS APRKNTNSVK YRLKFRFG Amino acid sequence of human FGF6(SEQ ID NO: 223) (NP 066276, which is hereby incorporated by reference in its entirety):   1 MALGQKLFIT MSRGAGRLQG TLWALVFLGI LVGMVVPSPA GTRANNTLLD SRGWGTLLSR  61 SRAGLAGEIA GVNWESGYLV GIKRQRRLYC NVGIGFHLQV LPDGRISGTH EENPYSLLEI 121 STVERGVVSL FGVRSALFVA MNSKGRLYAT PSFQEECKFR ETLLPNNYNA YESDLYQGTY 181 IALSKYGRVK RGSKVSPIMT VTHFLPRI Amino acid sequence of human FGF9(SEQ ID NO: 224) (GenBank accession no. NP 002001, which is hereby incorporated by reference in its entirety):   1 MAPLGEVGNY FGVQDAVPFG NVPVLPVDSP VLLSDHLGQS EAGGLPRGPA VTDLDHLKGI  61 LRRRQLYCRT GFHLEIFPNG TIQGTRKDHS RFGILEFISI AVGLVSIRGV DSGLYLGMNE 121 KGELYGSEKL TQECVFREQF EENWYNTYSS NLYKHVDTGR RYYVALNKDG TPREGTRTKR 181 HQKFTHFLPR PVDPDKVPEL YKDILSQS Amino acid sequence of human FGF16(SEQ ID NO: 225) (GenBank accession no. NP 003859, which is hereby incorporated by reference in its entirety):   1 MAEVGGVFAS LDWDLHGFSS SLGNVPLADS PGFLNERLGQ IEGKLQRGSP TDFAHLKGIL  61 RRRQLYCRTG FHLEIFPNGT VHGTRHDHSR FGILEFISLA VGLISIRGVD SGLYLGMNER 121 GELYGSKKLT RECVFREQFE ENWYNTYAST LYKHSDSERQ YYVALNKDGS PREGYRTKRH 181 QKFTHFLPRP VDPSKLPSMS RDLFHYR Amino acid sequence of human FGF20(SEQ ID NO: 226) (GenBank accession no. NP 062825, which is hereby incorporated by reference in its entirety):   1 MAPLAEVGGF LGGLEGLGQQ VGSHFLLPPA GERPPLLGER RSAAERSARG GPGAAQLAHL  61 HGILRRRQLY CRTGFHLQIL PDGSVQGTRQ DHSLFGILEF ISVAVGLVSI RGVDSGLYLG 121 MNDKGELYGS EKLTSECIFR EQFEENWYNT YSSNIYKHGD TGRRYFVALN KDGTPRDGAR 181 SKRHQKFTHF LPRPVDPERV PELYKDLLMY T 

It will be understood that the portion of the paracrine FGF according tothe present invention may be derived from a nucleotide sequence thatencodes human FGF4, FGF5, FGF6, FGF9, FGF16, and/or FGF20 having thenucleotide sequences shown in Table 6, or orthologs thereof.

TABLE 6 Human FGF4 gene coding sequence (1-206) (SEQ ID NO: 227) (GenBank accession no. NM_002007, which is hereby incorporated by reference in its entirety):  320                     A TGTCGGGGCC CGGGACGGCC GCGGTAGCGC TGCTCCCGGC  361 GGTCCTGCTG GCCTTGCTGG CGCCCTGGGC GGGCCGAGGG GGCGCCGCCG CACCCACTGC  421 ACCCAACGGC ACGCTGGAGG CCGAGCTGGA GCGCCGCTGG GAGAGCCTGG TGGCGCTCTC  481 GTTGGCGCGC CTGCCGGTGG CAGCGCAGCC CAAGGAGGCG GCCGTCCAGA GCGGCGCCGG  541 CGACTACCTG CTGGGCATCA AGCGGCTGCG GCGGCTCTAC TGCAACGTGG GCATCGGCTT  601 CCACCTCCAG GCGCTCCCCG ACGGCCGCAT CGGCGGCGCG CACGCGGACA CCCGCGACAG  661 CCTGCTGGAG CTCTCGCCCG TGGAGCGGGG CGTGGTGAGC ATCTTCGGCG TGGCCAGCCG  721 GTTCTTCGTG GCCATGAGCA GCAAGGGCAA GCTCTATGGC TCGCCCTTCT TCACCGATGA  781 GTGCACGTTC AAGGAGATTC TCCTTCCCAA CAACTACAAC GCCTACGAGT CCTACAAGTA  841 CCCCGGCATG TTCATCGCCC TGAGCAAGAA TGGGAAGACC AAGAAGGGGA ACCGAGTGTC  901 GCCCACCATG AAGGTCACCC ACTTCCTCCC CAGGCTGTGA Human FGF5 gene coding sequence (1-268) (SEQ ID NO: 228) (GenBank Accession No. NM_004464, which is hereby incorporated by reference in its entirety):  238                                                               ATG  241 AGCTTGTCCT TCCTCCTCCT CCTCTTCTTC AGCCACCTGA TCCTCAGCGC CTGGGCTCAC  301 GGGGAGAAGC GTCTCGCCCC CAAAGGGCAA CCCGGACCCG CTGCCACTGA TAGGAACCCT  361 AGAGGCTCCA GCAGCAGACA GAGCAGCAGT AGCGCTATGT CTTCCTCTTC TGCCTCCTCC  421 TCCCCCGCAG CTTCTCTGGG CAGCCAAGGA AGTGGCTTGG AGCAGAGCAG TTTCCAGTGG  481 AGCCCCTCGG GGCGCCGGAC CGGCAGCCTC TACTGCAGAG TGGGCATCGG TTTCCATCTG  541 CAGATCTACC CGGATGGCAA AGTCAATGGA TCCCACGAAG CCAATATGTT AAGTGTTTTG  601 GAAATATTTG CTGTGTCTCA GGGGATTGTA GGAATACGAG GAGTTTTCAG CAACAAATTT  661 TTAGCGATGT CAAAAAAAGG AAAACTCCAT GCAAGTGCCA AGTTCACAGA TGACTGCAAG  721 TTCAGGGAGC GTTTTCAAGA AAATAGCTAT AATACCTATG CCTCAGCAAT ACATAGAACT  781 GAAAAAACAG GGCGGGAGTG GTATGTGGCC CTGAATAAAA GAGGAAAAGC CAAACGAGGG  841 TGCAGCCCCC GGGTTAAACC CCAGCATATC TCTACCCATT TTCTGCCAAG ATTCAAGCAG  901 TCGGAGCAGC CAGAACTTTC TTTCACGGTT ACTGTTCCTG AAAAGAAAAA GCCACCTAGC  961 CCTATCAAGC CAAAGATTCC CCTTTCTGCA CCTCGGAAAA ATACCAACTC AGTGAAATAC 1021 AGACTCAAGT TTCGCTTTGG ATAA Human FGF6 gene coding sequence (1-208) (SEQ ID NO: 229) (NM_020996, which is hereby incorporated by reference in its entirety):   45                                                 ATGGCC CTGGGACAGA   61 AACTGTTCAT CACTATGTCC CGGGGAGCAG GACGTCTGCA GGGCACGCTG TGGGCTCTCG  121 TCTTCCTAGG CATCCTAGTG GGCATGGTGG TGCCCTCGCC TGCAGGCACC CGTGCCAACA  181 ACACGCTGCT GGACTCGAGG GGCTGGGGCA CCCTGCTGTC CAGGTCTCGC GCGGGGCTAG  241 CTGGAGAGAT TGCCGGGGTG AACTGGGAAA GTGGCTATTT GGTGGGGATC AAGCGGCAGC  301 GGAGGCTCTA CTGCAACGTG GGCATCGGCT TTCACCTCCA GGTGCTCCCC GACGGCCGGA  361 TCAGCGGGAC CCACGAGGAG AACCCCTACA GCCTGCTGGA AATTTCCACT GTGGAGCGAG  421 GCGTGGTGAG TCTCTTTGGA GTGAGAAGTG CCCTCTTCGT TGCCATGAAC AGTAAAGGAA  481 GATTGTACGC AACGCCCAGC TTCCAAGAAG AATGCAAGTT CAGAGAAACC CTCCTGCCCA  541 ACAATTACAA TGCCTACGAG TCAGACTTGT ACCAAGGGAC CTACATTGCC CTGAGCAAAT  601 ACGGACGGGT AAAGCGGGGC AGCAAGGTGT CCCCGATCAT GACTGTCACT CATTTCCTTC  661 CCAGGATCTA A Human FGF9 gene coding sequence (1-208)(SEQ ID NO: 230) (GenBank accession no. NM_002010, which is hereby incorporated by reference in its entirety):   838 ATG  841 GCTCCCTTAG GTGAAGTTGG GAACTATTTC GGTGTGCAGG ATGCGGTACC GTTTGGGAAT  901 GTGCCCGTGT TGCCGGTGGA CAGCCCGGTT TTGTTAAGTG ACCACCTGGG TCAGTCCGAA  961 GCAGGGGGGC TCCCCAGGGG ACCCGCAGTC ACGGACTTGG ATCATTTAAA GGGGATTCTC 1021 AGGCGGAGGC AGCTATACTG CAGGACTGGA TTTCACTTAG AAATCTTCCC CAATGGTACT 1081 ATCCAGGGAA CCAGGAAAGA CCACAGCCGA TTTGGCATTC TGGAATTTAT CAGTATAGCA 1141 GTGGGCCTGG TCAGCATTCG AGGCGTGGAC AGTGGACTCT ACCTCGGGAT GAATGAGAAG 1201 GGGGAGCTGT ATGGATCAGA AAAACTAACC CAAGAGTGTG TATTCAGAGA ACAGTTCGAA 1261 GAAAACTGGT ATAATACGTA CTCATCAAAC CTATATAAGC ACGTGGACAC TGGAAGGCGA 1321 TACTATGTTG CATTAAATAA AGATGGGACC CCGAGAGAAG GGACTAGGAC TAAACGGCAC 1381 CAGAAATTCA CACATTTTTT ACCTAGACCA GTGGACCCCG ACAAAGTACC TGAACTGTAT 1441 AAGGATATTC TAAGCCAAAG TTGA Human FGF16 gene coding sequence (1-207) (SEQ ID NO: 231) (GenBank accession no. NM_003868, which is hereby incorporated by reference in its entirety):    1 ATGGCAGAGG TGGGGGGCGT CTTCGCCTCC TTGGACTGGG ATCTACACGG CTTCTCCTCG   61 TCTCTGGGGA ACGTGCCCTT AGCTGACTCC CCAGGTTTCC TGAACGAGCG CCTGGGCCAA  121 ATCGAGGGGA AGCTGCAGCG TGGCTCACCC ACAGACTTCG CCCACCTGAA GGGGATCCTG  181 CGGCGCCGCC AGCTCTACTG CCGCACCGGC TTCCACCTGG AGATCTTCCC CAACGGCACG  241 GTGCACGGGA CCCGCCACGA CCACAGCCGC TTCGGAATCC TGGAGTTTAT CAGCCTGGCT  301 GTGGGGCTGA TCAGCATCCG GGGAGTGGAC TCTGGCCTGT ACCTAGGAAT GAATGAGCGA  361 GGAGAACTCT ATGGGTCGAA GAAACTCACA CGTGAATGTG TTTTCCGGGA ACAGTTTGAA  421 GAAAACTGGT ACAACACCTA TGCCTCAACC TTGTACAAAC ATTCGGACTC AGAGAGACAG  481 TATTACGTGG CCCTGAACAA AGATGGCTCA CCCCGGGAGG GATACAGGAC TAAACGACAC  541 CAGAAATTCA CTCACTTTTT ACCCAGGCCT GTAGATCCTT CTAAGTTGCC CTCCATGTCC  601 AGAGACCTCT TTCACTATAG GTAA Human FGF20 gene coding sequence (1-211) (SEQ ID NO: 232) (GenBank accession no. NM_019851, which is hereby incorporated by reference in its entirety):  134               ATGGCTC CCTTAGCCGA AGTCGGGGGC TTTCTGGGCG GCCTGGAGGG  181 CTTGGGCCAG CAGGTGGGTT CGCATTTCCT GTTGCCTCCT GCCGGGGAGC GGCCGCCGCT  241 GCTGGGCGAG CGCAGGAGCG CGGCGGAGCG GAGCGCGCGC GGCGGGCCGG GGGCTGCGCA  301 GCTGGCGCAC CTGCACGGCA TCCTGCGCCG CCGGCAGCTC TATTGCCGCA CCGGCTTCCA  361 CCTGCAGATC CTGCCCGACG GCAGCGTGCA GGGCACCCGG CAGGACCACA GCCTCTTCGG  421 TATCTTGGAA TTCATCAGTG TGGCAGTGGG ACTGGTCAGT ATTAGAGGTG TGGACAGTGG  481 TCTCTATCTT GGAATGAATG ACAAAGGAGA ACTCTATGGA TCAGAGAAAC TTACTTCCGA  541 ATGCATCTTT AGGGAGCAGT TTGAAGAGAA CTGGTATAAC ACCTATTCAT CTAACATATA  601 TAAACATGGA GACACTGGCC GCAGGTATTT TGTGGCACTT AACAAAGACG GAACTCCAAG  661 AGATGGCGCC AGGTCCAAGA GGCATCAGAA ATTTACACAT TTCTTACCTA GACCAGTGGA  721 TCCAGAAAGA GTTCCAGAAT TGTACAAGGA CCTACTGATG TACACTTGA 

As noted above, the chimeric protein includes a portion of a paracrineFGF coupled to a C-terminal region derived from an FGF19. FGF19 has beenshown to target and have effects on both adipocytes and hepatocytes. Forexample, mice harboring a FGF19 transgene, despite being on a high-fatdiet, show increased metabolic rates, increased lipid oxidation, a lowerrespiratory quotient and weight loss. Moreover, such mice showed lowerserum levels of leptin, insulin, cholesterol and triglycerides, andnormal levels of blood glucose despite the high-fat diet and withoutappetite diminishment (Tomlinson et al., “Transgenic Mice ExpressingHuman Fibroblast Growth Factor-19 Display Increased Metabolic Rate andDecreased Adiposity,” Endocrinology 143(5), 1741-1747 (2002), which ishereby incorporated by reference in its entirety). Obese mice thatlacked leptin but harbored a FGF19 transgene showed weight loss, loweredcholesterol and triglycerides, and did not develop diabetes. Obese,diabetic mice that lacked leptin, when injected with recombinant humanFGF19, showed reversal of their metabolic characteristics in the form ofweight loss and lowered blood glucose (Fu et al., “Fibroblast GrowthFactor 19 Increases Metabolic Rate and Reverses Dietary andLeptin-deficient Diabetes,” Endocrinology 145(6), 2594-2603 (2004),which is hereby incorporated by reference in its entirety).

In one embodiment of the present invention, FGF19 is human FGF19 and hasan amino acid sequence of SEQ ID NO: 233 (GenBank Accession No.NP_005108, which is hereby incorporated by reference in its entirety),or a portion thereof, as follows:

  1 MRSGCVVVHV WILAGLWLAV AGRPLAFSDA GPHVHYGWGD PIRLRHLYTS GPHGLSSCFL  61 RIRADGVVDC ARGQSAHSLL EIKAVALRTV AIKGVHSVRY LCMGADGKMQ GLLQYSEEDC 121 AFEEEIRPDG YNVYRSEKHR LPVSLSSAKQ RQLYKNRGFL PLSHFLPMLP MVPEEPEDLR 181 GHLESDMFSS PLETDSMDPF GLVTGLEAVR SPSFEK 

In one embodiment, the C-terminal portion of FGF19 of the chimericprotein of the present invention does not include any of residues 1 to168 of SEQ ID NO: 1. In certain embodiments of the present invention,the chimeric protein of the present invention does not include residuescorresponding to residues spanning residues 1 to 168 of SEQ ID NO:1. Inone embodiment, the C-terminal portion of FGF19 begins at a residuecorresponding to any one of residues 169, 197, or 204 of SEQ ID NO: 1.

In another embodiment, the C-terminal portion of FGF19 of the chimericprotein of the present invention comprises an amino acid sequencespanning residues corresponding to residues selected from the groupconsisting of from position 204 to 216 of SEQ ID NO: 1, from position197 to 216 of SEQ ID NO: 1, and from position 169 to 216 of SEQ IDNO: 1. In yet another embodiment, the C-terminal portion of FGF19 of thechimeric protein of the present invention comprises an amino acidsequence spanning residues of SEQ ID NO:1, which correspond to residues191 to 206 or 191 to 209 of SEQ ID NO: 1.

In one embodiment of the present invention, FGF19 or a portion thereofis from a mammalian FGF19. In one embodiment of the present invention,FGF19 or a portion thereof is from a vertebrate FGF19. In oneembodiment, FGF19 or a portion thereof is from a non-human vertebrateFGF19. It will be understood that this includes orthologs of humanFGF19, or a polypeptide or protein obtained from one species that is thefunctional counterpart of a polypeptide or protein from a differentspecies. In one embodiment, the C-terminal portion of FGF19 of thechimeric protein of the present invention is from human FGF19. In oneembodiment of the present invention, the C-terminal portion of FGF19 isfrom an ortholog of human FGF19 from gorilla gorilla, pan troglodytes,macaca mulatta, pongo abelii, nomascus leucogenys, callithrix jacchus,microcebus murinus, choloepus hoffmanni, ailuropoda melanoleuca, susscrofa, bos taurus, canis lupus familiaris, oryctolagus, pteropusvampyrus, tursiops truncates, myotis lucifugus, ornithorhynchusanatinus, monodelphis domestica, anolis carolinensis, ochotona princeps,cavia porcellus, tupaia belangeri, rattus norvegicus, mus musculus,gallus gallus, taeniopygia guttata, danio rerio, xenopus (silurana)tropicalis , otolemur garnettii, felis catus, pelodiscus sinensis,latimeria chalumnae, mustela putorius furo, takifugu rubripes, equuscaballus, oryzias latipes, xiphophorus maculatus, ictidomystridecemlineatus, gasterosteus aculeatus, oreochromis niloticus,meleagris gallopavo, papio anubis, saimiri boliviensis boliviensis,pteropus alecto, myotis davidii, tupaia chinensis, or heterocephalusglaber.

In other embodiments of the present invention, the portion of FGF19 ofthe chimeric protein of the present invention is from an ortholog ofhuman FGF19 having an amino acid sequence as shown in Table 7. Theportions of an ortholog of human FGF19 of a chimeric protein accordingto the present invention include portions corresponding to theabove-identified amino acid sequences of human FGF19. Correspondingportions may be determined by, for example, sequence analysis andstructural analysis. The high degree of FGF19 sequence conservationamong orthologs is shown in FIG. 12.

TABLE 7  Gorilla gorilla (gorilla) FGF19 (Ensembl Accession No. ENSGGOP00000021055, which is hereby incorporated by reference in its entirety) (SEQ ID NO: 234)   1 MRSGCVVVHV WILAGLWLAV AGRPLAFSDA GPHVHYGWGD PIRLRHLYTS GPHGLSSCFL  61 RIRADGVVDC ARGQSAHSLL EIKAVALRTV AIKGVHSVRY LCMGADGKMQ GLLQYSEEDC 121 AFEEEIRPDG YNVYRSEKHR LPVSLSSAKQ RQLYKNRGFL PLSHFLPMLP MVPEEPEDLR 181 GHLESDMFSS PLETDSMDPF GLVTGLEAVR SPSFEK Pan troglodytes (chimpanzee) FGF19 (Ensembl Accession No. ENSPIRP00000006877, which is hereby incorporated by reference in its entirety) (SEQ ID NO: 235)   1 MRNGCVVVHV WILAGLWLAV AGRPLAFSDA GRHVHYCWGD PIPLRHLYTS GPHGLSSCFL  61 RIPANCVMNC ARGQSAHSLL EIKAVALRTV AIKGVHSVRY LCMGADGKMQ GLLQYSEEDC 121 AFEEEIRPDG YNVYRSEKHR LPVSLSSAKQ RQLYKNRGFL PLSHFLPMLP MVPEEPEDLR 181 GHLESDMFSS PLETDSMDPF GLVTGLEAVR SPSFEK Macaca mulatta (Rhesus monkey) FGF19 (GenBank Accession No. XP 001100825, which is hereby incorporated by reference in its entirety) (SEQ ID NO: 236)   1 MRSGCVVVHA WILASLWLAV AGRPLAFSDA GPHVHYGWGD PIRLRHLYTS GPHGLSSCFL  61 RIRTDGVVDC ARGQSAHSLL EIKAVALRTV AIKGVHSVRY LCMGADGKMQ GLLQYSEEDC 121 AFEEEIRPDG YNVYRSEKHR LPVSLSSAKQ RQLYKNRGFL PLSHFLPMLP MAPEEPEDLR 181 GHLESDMFSS PLETDSMDPF GLVTGLEAVR SPSFEK Pongo abelii (Sumatran orangutan) FGF19 (GenBank Accession No. XP 002821459, which is hereby incorporated by reference in its entirety) (SEQ ID NO: 237)   1 MRSGCVVVHA WILAGLWLAV AGRPLAFSDS GPHVHYGWGD PIRLRHLYTS GPHGLSSCFL  61 RIRADGVVDC ARGQSAHSLL EIKAVALRTV AIKGVHSVRY LCMGADGKMQ GLLQYSEEDC 121 AFEEEIRPDG YNVYRSEKHR LPVSLSSAKQ RQLYKNRGFL PLSHFLPMLP MVPEEPEDLR 181 RHLESDMFSS PLETDSMDPF GLVTGLEAVR SPSFEK Nomascus leucogenys (Northern white-cheeked gibbon) FGF19 (Genbank Accession No. XP 003278071, which is hereby incorporated by reference in its entirety) (SEQ ID NO: 238)   1 MRSECVVVHA WILAGLWLAV AGRPLAFSDA GPHVHYGWGD PIRLRHLYTS GPHGLSSCFL  61 RIRADGVVDC ARGQSAHSLL EIKAVALRTV AIKGVHSVRY LCMGADGKMQ GLLQYSEEDC 121 AFEEEIRPDG YNVYRSEKHR LPVSLSSAKQ RQLYKNRGFL PLSHFLPMLP MVPEEPEDLR 181 GHLESDMFSS PLETDSMDPF GLVTGLEAVR SPSFEK Callithrix jacchus (white-tufted-ear marmoset) FGF19 (GenBank Accession No. XP 002763730, which is hereby incorporated by reference in its entirety) (SEQ ID NO: 239)   1 MWKATAGGQQ GQSEAQMSTC PHVPRPLWIA QSCLFSLQLQ YSEEDCAFEE EIRPDGYNVY  61 WSEKHRLPVS LSSAKQRQLY KKRGFLPLSH FLPMLPIAPE EPEDLRGHLE SDVFSSPLET 121 DSMDPFGLVT GLEAVNSPSF EK Microcebus murinus (mouse lemur) FGF19 (Ensembl Accession No. ENSMICP00000002788, which is hereby incorporated by reference in its entirety) (SEQ ID NO: 240)   1 MPSGQSGCVA ARALILAGLW LTAAGRPLAF SDAGPHVHYG WGEPIRLRHL YTAGPHGLSS  61 CFLRIRADGS VDCARGQSAH SLLEIRAVAL RTVAIKGVHS VRYLCMGADG RMQGLLRYSE 121 EDCAFEEEIR PDGYNVYRSE KHRLPVSLSS ARQRQLYKGR GFLPLSHFLP MLPVTPAETG 181 DLRDHLESDM FASPLETDSM DPFGIATRLG VVKSPSFQK Choloepus hoffmanni (sloth) FGF19 (Ensembl Accession No. ENSCHOP00000002044, which is hereby incorporated by reference in its entirety) (SEQ ID NO: 241) (partial amino acid sequence corresponding to human FGF19 residues 79 to 216)   1 LLEMKAVALR AVAIKGVHSA LYLCMNADGS LHGLPRYSAE DCAFEEEIRP DGYNVYWSRK  61 HGLPVSLSSA KQRQLYKGRG FLPLSHFLPM LPMTPAEPAD PGDDVESDMF SSPLETDSMD 121 PFGIASRLEL VNSPSFQT Ailuropoda melanoleuca (giant panda) FGF19 (GenBank Accession No. XP 002927952, which is hereby incorporated by reference in its entirety) (SEQ ID NO: 242) (partial amino acid sequence corresponding to human FGF19 residues 12 to 216) 124 VLAGLCL AVAGRPLAFS DAGPHVHYGW GEPIRLRHLY TAGPHGLSSC FLRIRADGGV 181 DCARGQSAHS LVEIRAVALR TVAIKGVHSV RYLCMGADGR MQGLPQYSAG DCAFEEEIRP 241 DGYNVYRSKK HRLPVSLSGA KQRQLYKDRG FLPLSHFLPM LPGSPAEPRD LQDHAESDGF 301 SAPLETDSMD PFGIATKMGL VKSPSFQK Sus scrofa (pig) FGF19 (Ensembl Accession No. EN555CP00000013682, which is hereby incorporated by reference in its entirety) (SEQ ID NO: 243) 1 MRSAPSRCAV VRALVLAGLW LAAAGRPLAF SDAGPHVHYG WGESVRLRHL YTASPHGVSS  61 CFLRIHSDGP VDCAPGQSAH SLMEIRAVAL STVAIKGERS RYLCMGADGK MQGQTQYSDE 121 DCAFEEEIRP DGYNVYWSKK HHLPVSLSSA RQRQLYKGRG FLPLSHFLPM LSTLPAEPED 181 LQDPFKSDLF SLPLETDSMD PFRIAAKLGA VKSPSFYK Bos taurus (bovine) FGF19 (GenBank Accession No. XP 599739, which is hereby incorporated by reference in its entirety) (SEQ ID NO: 244) 136 MRSAP SRCAVARALV LAGLWLAAAG RPLAFSDAGP HVHYGWGESV 181 RLRHLYTAGP QGLYSCFLRI HSDGAVDCAQ VQSAHSLMEI RAVALSTVAI KGERSVLYLC 241 MDADGKMQGL TQYSAEDCAF EEEIRPDGYN VYWSRKHHLP VSLSSSRQRQ LFKSRGFLPL 301 SHFLPMLSTI PAEPEDLQEP LKPDFFLPLK TDSMDPFGLA TKLGSVKSPS FYN Canis lupus familiaris (dog) FGF19 (GenBank Accession No. XP 540802, which is hereby incorporated by reference in its entirety) (SEQ ID NO: 245) (partial amino acid sequence corresponding to human FGF19 residues 25 to 216)   1 LAFSDAGPHV HSFWGEPIRL RHLYTAGPHG LSSCFLRIRA DGGVDCARGQ SAHSLMEMRA  61 VALRTVAIKG VHSGRYLCMG ADGRMQGLPQ YSAGDCTFEE EIRPDGYNVY WSKKHHLPIS 121 LSSAKQRQLY KGRGFLPLSH FLPILPGSPT EPRDLEDHVE SDGFSASLET DSMDPFGIAT 181 KIGLVKSPSF QK Oryctolagus cuniculus (rabbit) FGF19 (GenBank Accession No. XP 002724495, which is hereby incorporated by reference in its entirety) (SEQ ID NO: 246)   1 MRRAPSGGAA ARALVLAGLW LAAAARPLAL SDAGPHLHYG WGEPVRLRHL YATSAHGVSH  61 CFLRIRADGA VDCERSQSAH SLLEIRAVAL RTVAFKGVHS SRYLCMGADG RMRGQLQYSE 121 EDCAFQEEIS SGYNVYRSTT HHLPVSLSSA KQRHLYKTRG FLPLSHFLPV LPLASEETAA 181 LGDHPEADLF SPPLETDSMD PFGMATKLGP VKSPSFQK Pteropus vampyrus (megabat) FGF19 (Ensembl Accession No. ENSPVAP00000009339, which is hereby incorporated by reference in its entirety) (SEQ ID NO: 247)   1 MRSPCAVARA LVLAGLWLAS AAGPLALSDA GPHVHYGWGE AIRLRHLYTA GPHGPSSCFL  61 RIRADGAVDC ARGQSAHSLV EIRAVALRNV AIKGVHSVRY LCMGADGRML GLLQYSADDC 121 AFEEEIRPDG YNVYHSKKHH LPVSLSSAKQ RQLYKDRGFL PLSHFLPMLP RSPTEPENFE 181 DHLEADTFSS LETDDMDPFG IASKLGLEES PSFQK Tursiops truncatus (dolphin) FGF19 (Ensembl Accession No. ENSTIRP00000000061, which is hereby incorporated by reference in its entirety) (SEQ ID NO: 248)   1 MRSAPSRCAV ARALVLAGLW LAAAGRPLAF SDAGPHVHYG WGESVRLRHL YTAGPQGLSS  61 CFLRIHSDGA VDCAPVQSAH SLMEIRAVAL STVAIKGERS VLYLCMGADG KMQGLSQYSA 121 EDCAFEEEIR PDGYNVYWSK KHHLPVSLSS ARQRQLFKGR GFLPLSHFLP MLSTIPTEPD 181 EIQDHLKPDL FALPLKTDSM DPFGLATKLG VVKSPSFYK Myotis lucifugus (microbat) FGF19 (Ensembl Accession No. ENSMLUP00000002279, which is hereby incorporated by reference in its entirety) (SEQ ID NO: 249)   1 MQSAWSRRVV ARALVLASLG LASAGGPLGL SDAGPHVHYG WGESIRLRHL YTSGPHGPSS  61 CFLRIRADGA VDCARGQSAH SLVEIRAVAL RKVAIKGVHS ALYLCMGGDG RMLGLPQFSP 121 EDCAFEEEIR PDGYNVYRSQ KHQLPVSLSS ARQRQLFKAR GFLPLSHFLP MLPSSPAGPV 181 PRERPSEPDE FSSPLETDSM DPFGIANNLR LVRSPSFQE Ornithorhynchus anatinus (platypus) FGF19 (GenBank Accession No. XP 001506714, which is hereby incorporated by reference in its entirety)(SEQ ID NO: 250) (partial amino acid sequence corresponding to human FGF19 residues 79 to 216)   1 MLSCVVLPSL LEIKAVAVRT VAIKGVHISR YLCMEEDGKT PWARLLEIKA VAVRTVAIKG  61 VHSSRYLCME EDGKLHGQIW YSAEDCAFEE EIRPDGYNVY KSKKYGVPVS LSSAKQRQQF 121 KGRDFLPLSR FLPMINTVPV EPAEFGDYAD YFESDIFSSP LETDSMDPFR IAPKLSPVKS 181 PSFQK  Monodelphis domestica (opossum) FGF19 (GenBank Accession No. XP 001506714, which is hereby incorporated by reference in its entirety) (SEQ ID NO: 251)   1 MAQLLAPLLT LAALWLAPTA RARPLVDAGP HVYYGWGEPI RLRHLYTANR HGLASFSFLR  61 IHRDGRVDGS RSQSALSLLE IKAVALRMVA IKGVHSSRYL CMGDAGKLQG SVRFSAEDCT 121 FEEQIRPDGY NVYQSPKYNL PVSLCTDKQR QQAHGKEHLP LSHFLPMINA IPLEAEEPEG 181 PRMLAAPLET DSMDPFGLTS KLLPVKSPSF QK Anolis carolinensis (anole lizard) FGF19 (GenBank Accession No. XP 003214715, which is hereby incorporated by reference in its entirety) (SEQ ID NO: 252)   1 MCRRALPLLG ALLGLAAVAS RALPLTDAGP HVSYGWGEPV RLRHLYTAGR QGLFSQFLRI  61 HADGRVDGAG SQNRQSLLEI RAVSLRAVAL KGVHSSRYLC MEEDGRLRGM LRYSAEDCSF 121 EEEMRPDGYN IYKSKKYGVL VSLSNARQRQ QFKGKDFLPL SHFLPMINTV PVESADFGEY 181 GDTRQHYESD IFSSRLETDS MDPFGLTSEV SSVQSPSFGK Ochotona princeps (pika) FGF19 (Ensembl Accession No. ENSOPRP00000009838, which is hereby incorporated by reference in its entirety) (SEQ ID NO: 253) (partial amino acid sequence corresponding to human FGF19 residues 12 to 77 and 113 to 216)   1 VRSRGAMARA LVLATLWLAA TGRPLALSDA GPHLHYGWGE PIRLRHLYAT SAHGLSHCFL  61 RIRIDGIVDC ERSQSAH LQYSEEDC 121 AFEEEISSGY NVYRSRRYQL PVSLGSARQR QLQRSRGFLP LSHFLPVLPA ASEEVAAPAD 181 HPQADPFSPL ETDSMDPFGM ATKRGLVKSP SFQK Cavia porcellus (guinea pig) FGF19 (Ensembl Accession No. ENSCPOP00000007325, which is hereby incorporated by reference in its entirety) (SEQ ID NO: 254)   1 MWSAPSGCVV IRALVLAGLW LAVAGRPLAR RSLALSDQGP HLYYGWDQPI RLRHLYAAGP  61 YGRSRCFLRI HTDGAVDCVE EQSEHCLLEI RAVALETVAI KDINSVRYLC MGPDGRMRGL 121 PWYSEEDCAF KEEISYPGYS VYRSQKHHLP IVLSSVKQRQ QYQSKGVVPL SYFLPMLPKA 181 SVEPSDEEES SVFSLPLKTD SMDPFGMASE IGLVKSPSFQ K Tupaia belangeri (tree shrew) FGF19 (Ensembl Accession No. ENSTBEP00000000264, which is hereby incorporated by reference in its entirety) (SEQ ID NO: 255) (partial amino acid sequence corresponding to human FGF19 (residues 1 to 112 and 136 to 216)   1 MRRTPSGFAV ARVLFLGSLW LAAAGSPLAL SDAGPHVNYG WDESIRLRHL YTASPHGSTS  61 CFLRIRDDGS VDCARGQSLH SLLEIKAVAL QTVAIKGVYS VRYLCMDADG RMQGL 121 ST KHGLPVSLSS AKQRQLLTVR GFPSLPHFLL MMAKTSAGPG 181 NPRDHPGSNT FSLPLETDSM DPFGMTTRHG LVKSPSFQN Rattus norvegicus (Norway rat) FGF15 (GenBank Accession No. NP 570109, which is hereby incorporated by reference in its entirety) (SEQ ID NO: 256)   1 MARKWSGRIV ARALVLATLW LAVSGRPLVQ QSQSVSDEGP LFLYGWGKIT RLQYLYSAGP  61 YVSNCFLRIR SDGSVDCEED QNERNLLEFR AVALKTIAIK DVSSVRYLCM SADGKIYGLI 121 RYSEEDCTFR EEMDCLGYNQ YRSMKHHLHI IFIKAKPREQ LQGQKPSNFI PIFHRSFFES 181 TDQLRSKMFS LPLESDSMDP FRMVEDVDHL VKSPSFQK Mus musculus (house mouse) FGF15 (GenBank Accession No. NP 032029, which is hereby incorporated by reference in its entirety) (SEQ ID NO: 257) 1 MARKWNGRAV ARALVLATLW LAVSGRPLAQ QSQSVSDEDP LFLYGWGKIT RLQYLYSAGP  61 YVSNCFLRIR SDGSVDCEED QNERNLLEFR AVALKTIAIK DVSSVRYLCM SADGKIYGLI 121 RYSEEDCTFR EEMDCLGYNQ YRSMKHHLHI IFIQAKPREQ LQDQKPSNFI PVFHRSFFET 181 GDQLRSKMFS LPLESDSMDP FRMVEDVDHL VKSPSFQK Gallus gallus (chicken) FGF19 (GenBank Accession No. NP 990005, which is hereby incorporated by reference in its entirety) (SEQ ID NO: 258)   1 MGPARPAAPG AALALLGIAA AAAAARSLPL PDVGGPHVNY GWGEPIRLRH LLHRPGKHGL  61 FSCFLRIGGD GRVDAVGSQS PQSLLEIRAV AVRTVAIKGV QSSRYLCMDE AGRLHGQLSY 121 SIEDCSFEEE IRPDGYNVYK SKKYGISVSL SSAKQRQQFK GKDFLPLSHF LPMINTVPVE 181 VTDFGEYGDY SQAFEPEVYS SPLETDSMDP FGITSKLSPV KSPSFQK Taeniopygia guttata (zebra finch) FGF19 (GenBank Accession No. XP 002194493, which is hereby incorporated by reference in its entirety) (SEQ ID NO: 259)   1 MVIISNLYLM QNDVMMNMRR APLRVHAARS SATPASALPL PPPDAGPHLK YGWGEPIRLR  61 HLYTASKHGL FSCFLRIGAD GRVDAAGSQS PQSLLEIRAV AVRTVAIKGV QSSRYLCMDE 121 AGRLHGQLRN STEDCSFEEE IRPDGYNVYR SKKHGISVSL SSAKQRQQFK GKDFLPLSHF 181 LPMINTVPME SADFGEYGDY SQAFEAEAFS SPLETDSMDP FGIASKLSLV KSPSFQN Danio rerio (zebrafish) FGF19 (GenBank Accession No. NP 001012246, which is hereby incorporated by reference in its entirety) (SEQ ID NO: 260)   1 MLLLLFVTVC GSIGVESLPL PDSGPHLAND WSEAVRLRHL YAARHGLHLQ INTDGEIIGS  61 TCKARTVSLM EIWPVDTGCV AIKGVASSRF LCMERLGNLY GSHIYTKEDC SFLERILPDG 121 YNVYFSSKHG ALVTLSGAKN KLHSNDGTSA SQFLPMINTL SEEHTKQHSG EQHSSVNHGQ 181 DHQLGLEIDS MDPFGKISQI VIQSPSFNKR Xenopus (Silurana) tropicalis (western clawed frog) FGF19 (GenBank Accession No. NP 001136297, which is hereby incorporated by reference in its entirety) (SEQ ID NO: 261)   1 MWKTLPWILV PMMVAVLYFL GGAESLPLFD AGPHMQNGWG ESIRIRHLYT ARRFGHDSYY  61 LRIHEDGRVD GDRQQSMHSL LEIRAIAVGI VAIKGYRSSL YLCMGSEGKL YGMHSYSQDD 121 CSFEEELLPD GYNMYKSRKH GVAVSLSKEK QKQQYKGKGY LPLSHFLPVI SWVPMEPTGD 181 VEDDIYRFPF NTDIKSVIDS LDTLGLMDFS SYHKK Otolemur garnettii (bushbaby) FGF19 (Ensembl Accession No. EN50GAP00000017975, which is hereby incorporated by reference in its entirety) (SEQ ID NO: 262)   1 MPSGLRGRVV AGALALASFW LAVAGRPLAF SDAGPHVHYG WGEPIRLRHL YTAGPHGLSS  61 CFLRVRTDGA VDCARGQSAH SLLEIRAVAL RTVAIKGVHS ARYLCMGADG RMQGLPQYSE 121 EDCAFEEEIR PDGYNVYWSE KHRLPVSLSS ARQRQLYKGR GFLPLSHFLP MLPVTPAEPG 181 DLRDHLESDM FSLPLETDSM DPFGIATRLG VVKSPSFQK Felis catus (cat) FGF19 (Ensembl Accession No. ENSFCAP00000022548, which is hereby incorporated by reference in its entirety)(SEQ ID NO: 263)   1 MRSAPSQCAV TRALVLAGLW LAAAGRPLAF SDAGPHVHYG WGEPIRLRHL YTAGPHGLSS  61 CFLRIRADGG VDCARSQSAH SLVEIRAVAL RTVAIKGVHS VRYLCMGADG RMQGLLQYSA 121 GDCAFQEEIR PDGYNVYRSE KHRLPVSLSS AIQRQLYKGR GFLPLSHFLP MLPGSPAEPR 181 DLQDHVESER FSSPLETDSM DPFGIATKMG LVKSPSFQK Pelodiscus sinensis (Chinese softshell turtle) FGF19 (Ensembl Accession No. ENSPSIP00000010374, which is hereby incorporated by reference in its entirety) (SEQ ID NO: 264)   1 MWRSLCKSHT SLALLGLCFA VVVRSLPFSD AGPHVNYGWG EPIRLRHLYT ASRHGLFNYF  61 LRISSDGKVD GTSIQSPHSL LEIRAVAVRT VAIKGVHSSR YLCMEEDGKL HGLLRYSTED 121 CSFEEEIRPD GYNVYKSKKY GISVSLSSAK QRQQFKGKDF LPLSHFLPMI NTVPVESMDF 181 GEYGDYSHTF ESDLFSSPLE TDSMDPFGIT SKISPVKSPS FQK Latimeria chalumnae (coelacanth) FGF19 (Ensembl Accession No. ENSLACP00000014596, which is hereby incorporated by reference in its entirety) (SEQ ID NO: 265)   1 MLQALYNLCT ALVLFKLPFA MVGYTLPSAN EGPHLNYDWG ESVRLKHLYT SSKHGLISYF  61 LQINDDGKVD GTTTRSCYSL LEIKSVGPGV LAIKGIQSSR YLCVEKDGKL HGSRTYSADD 121 CSFKEDILPD GYTIYVSKKH GSVVNLSNHK QKRQRNRRTL PPFSQFLPLM DTIRVECMNC 181 GEHCDDNLHD ELETGLSMDP FESTSKKSFQ SPSFHNR Mustela putorius furo (ferret) FGF19 (Ensembl Accession No. ENSMPUP00000004571, which is hereby incorporated by reference in its entirety) (SEQ ID NO: 266)   1 MRSAASRCAV ARALVLAGLW LAAAGRPLAF SDAGPHVHYG WGEPIRLRHL YTAGPHGLSS  61 CFLRIRADGG VDCARGQSAH SLVEIRAVAL RTVAIKGVYS DRYLCMGADG RMQGLPQYSA 121 GDCAFEEEIR PDGYNVYRSK KHRLPVSLSS AKQRQLYKDR GFLPLSHFLP MLPGSLAEPR 181 DLQDHVEADG FSAPLETDSM DPFGIATKMG LVKSPSFQK Takifugu rubripes (fugu) FGF19 (Ensembl Accession No. ENSTRUP00000007110, which is hereby incorporated by reference in its entirety) (SEQ ID NO: 267)   1 SSTRISGNMV LLMLPITVAN LFLCAGVLSL PLLDQGSHFP QGWEQVVRFR HLYAASAGLH  61 LLITEEGSIQ GSADPTLYSL MEIRPVDPGC VVIRGAATTR FLCIEGAGRL YSSQTYSKDD 121 CTFREQILAD GYSVYRSVGH GALVSLGNYR QQLRGEDWSV PTLAQFLPRI SSLDQDFKAA 181 LDETEKPEQT APQRSEPVDM VDSFGKLSQI IHSPSFHK Equus caballus (horse) FGF19 (Ensembl Accession No. ENSECAP00000017705, which is hereby incorporated by reference in its entirety) (SEQ ID NO: 268); partial sequence corresponding to human FGF19 residues 20 to 113   1 AAGRPLALSD AGPHVHYGWG EPIRLRHLYT AGPHGLSSCF LRIRADGAVD CARGQSAHSL  61 VEIRAVALRT VAIKGVHSVR YLCMGADGRM QGLV Oryzias latipes (medaka) FGF19 (Ensembl Accession No. ENSORLP00000000352, which is hereby incorporated by reference in its entirety) (SEQ ID NO: 269)   1 TMLLIVVTIS TMVFSDSGVS SMPLSDHGPH ITHSWSQVVR LRHLYAVKPG QHVQIREDGH  61 IHGSAEQTLN SLLEIRPVAP GRVVFRGVAT SRFLCMESDG RLFSSHTFDK DNCVFREQIL 121 ADGYNIYISD QHGTLLSLGN HRQRQQGLDR DVPALAQFLP RISTLQQGVY PVPDPPHQMR 181 TMQTEKTLDA TDIFGQLSKI IHSPSFNKR Xiphophorus maculatus (platyfish) FGF19 (Ensembl Accession No. ENSXMAP00000001516, which is hereby incorporated by reference in its entirety) (SEQ ID NO: 270)   1 MFVFILCIAG ELFTLGVFCM PMMDQGPLVT HGWGQVVRHR HLYAAKPGLH LLISEDGQIH  61 GSADQTLYSL LEIQPVGPGR VVIKGVATTR FLCMESDGRL YSTETYSRAD CTFREQIQAD 121 GYNVYTSDSH GALLSLGNNQ QRHSGSDRGV PALARFLPRL NTLQQAVPTE PDVPDQLSPE 181 KVQQTVDMVA SFGKLSHIIH SPSFHKR Ictidomys tridecemlineatus (squirrel) FGF19 (Ensembl Accession No. ENSSTOP00000021639, which is hereby incorporated by reference in its entirety) (SEQ ID NO: 271)   1 MRSAPSGRAL ARALVLASLW LAVAGRPLAR RSLALSDQGP HLYYGWDQPI RLRHLYAAGP  61 YGFSNCFLRI RTDGAVDCEE KQSERSLMEI RAVALETVAI KDINSVRYLC MGADGRIQGL 121 PRYSEEECTF KEEISYDGYN VYRSQKYHLP VVLSSAKQRQ LYQSKGVVPL SYFLPMLPLA 181 SAETRDRLES DVFSLPLETD SMDPFGMASE VGLKSPSFQK Gasterosteus aculeatus (stickleback) FGF19 (Ensembl Accession No. ENSGACP00000018732, which is hereby incorporated by reference in its entirety) (SEQ ID NO: 272)   1 MLLLLVPAYV ASVFLALGVV CLPLTDQGLH MADDWGQSVR LKHLYAASPG LHLLIGEDGR  61 IQGSAQQSPY SLLEISAVDP GCVVIRGVAT ARFLCIEGDG RLYSSDTYSR DDCTFREQIL 121 PDGYSVYVSH GHGALLSLGN HRQRLQGRDH GVPALAQFLP RVSTMDQASA PDAPGQTATE 181 TEEPVDSFGK LSQIIHSPSF HER Oreochromis niloticus (tilapia) FGF19 (Ensembl Accession No. EN50NIP00000022796, which is hereby incorporated by reference in its entirety) (SEQ ID NO: 273)   1 MLLLLIVSIV NMLFGVGMVC MPLSDNGPHI AHGWAQVVRL RHLYATRPGM HLLISEGGQI  61 RGSAVQTLHS LMEIRPVGPG RVVIRGVATA RFLCIEDDGT LYSSHAYSRE DCIFREQILP 121 DGYNIYISDR HGVLLSLGNH RQRLQGLDRG DPALAQFLPR ISTLNQIPSP GANIGDHMKV 181 AKTEEPVDTI DSFGKFSQII DSPSFHKR Meleagris gallopavo (turkey) FGF19 (Ensembl Accession No. ENSMGAP00000010265, which is hereby incorporated by reference in its entirety) (SEQ ID NO: 274); partial sequence corresponding to human FGF19 residues 71 to 216   1 VGNQSPQSIL EITAVDVGIV AIKGLFSGRY LAMNKRGRLY ASLSYSIEDC SFEEEIRPDG  61 YNVYKSKKYG ISVSLSSAKQ RQQFKGKDFL PLSHFLPMIN TVPVEVTDFG EYGDYSQAFE 121 PEVYSSPLET DSMDPFGITS KLSPVKSPSF QK Papio anubis (olive baboon) FGF19 (GenBank Accession No. XP 003909471, which is hereby incorporated by reference in its entirety) (SEQ ID NO: 275)   1 MRSGCVVVHA WILASLWLAV AGRPLAFSDA GPHVHYGWGD PIRLRHLYTS GPHGLSSCFL  61 RIRTDGVVDC ARGQSAHSLL EIKAVALRTV AIKGVHSVRY LCMGADGKMQ GLLQYSEEDC 121 AFEEEIRPDG YNVYRSQKHR LPVSLSSAKQ RQLYKNRGFL PLSHFLPMLP MAPEEPEDLR 181 GPLESDMFSS PLETDSMDPF GLVTGLEAVR SPSFEK Saimiri boliviensis boliviensis (Bolivian squirrel monkey) FGF19 (GenBank Accession No. XP 003941214, which is hereby incorporated by reference in its entirety) (SEQ ID NO: 276)   1 MRSGCVVVHA WILAGLWLAV VGRPLAFSDA GPHVHYGWGD PIRLRHLYTS SPHGLSSCFL  61 RIRSDGVVDC ARGQSAHSLL EIKAVALRTV AIKGVHSSRY LCMGADGRLQ GLFQYSEEDC 121 AFEEEIRPDG YNVYLSEKHR LPVSLSSAKQ RQLYKKRGFL PLSHFLPMLP RAPEEPDDLR 181 GHLESDVFSS PLETDSMDPF GLVTGLEAVN SPSFEK Pteropus alecto (black flying fox) FGF19 (GenBank Accession No. ELK13233, which is hereby incorporated by reference in its entirety) (SEQ ID NO: 277)   1 MRSPCAVARA LVLAGLWLAS AAGPLALSDA GPHVHYGWGE AIRLRHLYTA GPHGPSSCFL  61 RIRADGAVDC ARGQSAHSLV EIRAVALRNV AIKGVHSVRY LCMGADGRML GLLQYSADDC 121 AFEEEIRPDG YNVYHSKKHH LPVSLSSAKQ RQLYKDRGFL PLSHFLPMLP RSPTEPENFE 181 DHLEADTFSS PLETDDMDPF GIASKLGLEE SPSFQK Myotis davidii (David's myotis) FGF19 (GenBank Accession No. ELK24234, which is hereby incorporated by reference in its entirety) (SEQ ID NO: 278)   1 MSGQNSGRHG SRPGLDEEPE PGPLELRALG STRADPQLCD FLENHFLGYT CLELDICLAT  61 YLGVSHWGES IRLRHLYTSG PHGPSSCFLR IRVDGAVDCA RGQSAHSLVE IRAVALRKVA 121 IKGVHSALYL CMEGDGRMRG LPQFSPEDCA FEEEIRPDGY NVYRSQKHQL PVSLSSARQR 181 QLFKARGFLP LSHFLPMLPS SPAEPVHRER PLEPDAFSSP LETDSMDPFG IANNLRLVKS 241 PSFQK Tupaia chinensis (Chinese tree shrew) FGF19 (GenBank Accession No. ELW64990, which is hereby incorporated by reference in its entirety) (SEQ ID NO: 279); residues 1-257, excluding 13-19   1 MRRTWSGFAV AT R AGSPLALADA GPHVNYGWDE SIRLRHLYTA SLHGSTSCFL  61 RIRDDGSVGC ARGQSMHSLL EIKAVALQTV AIKGVYSVRY LCMDTDGRMQ GLPQYSEEDC 121 TFEEEIRSDG HNVYRSKKHG LPVSLSSAKQ RQLYKGRGFL SLSHFLLMMP KTSAGPGNPR 181 DQRNPRDQRD PNTFSLPLET DSMDPFGMTT RHGLLLDSCC ASLVLLNIST DGEFSPYGNI 241 LRPSFRFKLF KMKKVTN Heterocephalus glaber (naked mole-rat) FGF19 (GenBank Accession No. EHB12332, which is hereby incorporated by reference in its entirety) (SEQ ID NO: 280)   1 MRFSKSTCGF FNHQRLQALW LSLSSVKWVL DAAVEGRPIR LRHLYAAGPY GRSRCFLRIH  61 TDGAVDCVEE QSEHCLLEIR AVALETVAIK DINSVRYLCM GPDGRMQGLP WYSEEDCAFK 121 EEISYPGYSV YRSQKHHLPI VLSSVKQRQQ YQSKGVVPLS YFLPMLPKAS VEPGDEEESA 181 FSLPLKTDSM DPFGMASEIG LAKSPSFQK 

In one embodiment, a C-terminal portion of FGF19 of the chimeric proteinof the present invention comprises the conserved amino acid sequenceTGLEAV(R/N)SPSFEK (SEQ ID NO: 281). In one embodiment, a C-terminalportion of FGF19 comprises the conserved amino acid sequenceMDPFGLVTGLEAV(R/N)SPSFEK (SEQ ID NO: 282). In one embodiment, theC-terminal portion of FGF19 of the chimeric protein of the presentinvention comprises the conserved amino acid sequenceLP(M/I)(V/A)PEEPEDLR(G/R) HLESD(M/V)FSSPLETDSMDPFGLVTGLEAV(R/N)SPSFEK(SEQ ID NO: 283).

In one embodiment, the C-terminal portion of FGF19 of the chimericprotein of the present invention consists of an amino acid sequenceselected from the group consisting of TGLEAV(R/N)SPSFEK (SEQ ID NO:281); MDPFGLVTGLEAV(R/N) SPSFEK (SEQ ID NO: 282); andLP(M/I)(V/A)PEEPEDLR(G/R)HLESD(M/V)FSS PLETDSMDPFGLVTGLEAV(R/N)SPSFEK(SEQ ID NO: 283).

In certain embodiments according to the present invention, theC-terminal portion of FGF19 of the chimeric protein of the presentinvention includes a polypeptide sequence that has at least 80%, atleast 85%, at least 90%, at least 95%, at least 97%, or at least 99%amino acid sequence identity to the amino acid sequences of any ofTGLEAV(R/N)SPSFEK (SEQ ID NO: 281); MDPFGLVTGLEAV(R/N)SPSFEK (SEQ ID NO:282); or LP(M/I)(V/A)PEEPEDLR(G/R)HLESD(M/V)FSSPLETDSMDPFGLVTGLEAV(R/N)SPSFEK (SEQ ID NO: 283). In certain embodiments according to thepresent invention, the C-terminal portion of FGF19 of the chimericprotein of the present invention includes a polypeptide sequence thathas at least 80%, at least 85%, at least 90%, at least 95%, at least97%, or at least 99% amino acid sequence homology to the amino acidsequences of any of TGLEAV(R/N)SPSFEK (SEQ ID NO: 281);MDPFGLVTGLEAV(R/N)SPSFEK (SEQ ID NO: 282); or LP(M/I)(V/A)PEEPEDLR(G/R)HLESD(M/V)FSSPLETDSMDPFGLVTGLEAV(R/N)SPSFEK (SEQ ID NO: 283).

It will be understood that the portion from FGF19 of the chimericprotein of the present invention may be from a nucleotide sequence thatencodes an FGF19 protein (e.g., those encoding orthologs) from a mammalor even a non-mammalian species. For example, a nucleotide sequenceencoding a mammalian or non-mammalian FGF19 protein according to thepresent invention may include, but is not limited to, those FGF-encodingnucleotide sequences shown in Table 8.

TABLE 8 Human FGF19 gene coding sequence (1-216)(SEQ ID NO: 284) (GenBank Accession No. NM_005117, which is hereby incorporated by reference in its entirety)  464    ATGCGGA GCGGGTGTGT GGTGGTCCAC GTATGGATCC TGGCCGGCCT CTGGCTGGCC  521 GTGGCCGGGC GCCCCCTCGC CTTCTCGGAC GCGGGGCCCC ACGTGCACTA CGGCTGGGGC  581 GACCCCATCC GCCTGCGGCA CCTGTACACC TCCGGCCCCC ACGGGCTCTC CAGCTGCTTC  641 CTGCGCATCC GTGCCGACGG CGTCGTGGAC TGCGCGCGGG GCCAGAGCGC GCACAGTTTG  701 CTGGAGATCA AGGCAGTCGC TCTGCGGACC GTGGCCATCA AGGGCGTGCA CAGCGTGCGG  761 TACCTCTGCA TGGGCGCCGA CGGCAAGATG CAGGGGCTGC TTCAGTACTC GGAGGAAGAC  821 TGTGCTTTCG AGGAGGAGAT CCGCCCAGAT GGCTACAATG TGTACCGATC CGAGAAGCAC  881 CGCCTCCCGG TCTCCCTGAG CAGTGCCAAA CAGCGGCAGC TGTACAAGAA CAGAGGCTTT  941 CTTCCACTCT CTCATTTCCT GCCCATGCTG CCCATGGTCC CAGAGGAGCC TGAGGACCTC 1001 AGGGGCCACT TGGAATCTGA CATGTTCTCT TCGCCCCTGG AGACCGACAG CATGGACCCA 1061 TTTGGGCTTG TCACCGGACT GGAGGCCGTG AGGAGTCCCA GCTTTGAGAA GTAA Gorilla FGF19 gene coding sequence (1-216) (SEQ ID NO: 285) (Ensembl Accession No. ENSGGOT00000028361, which is hereby incorporated by reference in its entirety)  463   ATGCGGAG CGGGTGTGTG GTGGTCCACG TCTGGATCCT GGCCGGCCTC TGGCTGGCCG  521 TGGCCGGGCG CCCCCTCGCC TTCTCGGACG CGGGGCCCCA CGTGCACTAC GGCTGGGGCG  581 ACCCCATCCG CCTGCGGCAC CTGTACACCT CCGGCCCCCA CGGGCTCTCC AGCTGCTTCC  641 TGCGCATCCG TGCCGACGGC GTCGTGGACT GCGCGCGGGG CCAGAGCGCG CACAGTTTGC  701 TGGAGATCAA GGCAGTCGCT CTGCGGACCG TGGCCATCAA GGGCGTGCAC AGCGTGCGGT  761 ACCTCTGCAT GGGCGCCGAC GGCAAGATGC AGGGGCTGCT TCAGTACTCG GAGGAAGACT  821 GTGCTTTCGA GGAGGAGATC CGCCCAGATG GCTACAATGT GTACCGATCT GAGAAGCACC  881 GCCTCCCGGT CTCCCTGAGC AGTGCCAAAC AGCGGCAGCT GTACAAGAAC AGAGGCTTTC  941 TTCCGCTCTC TCATTTCCTG CCCATGCTGC CCATGGTCCC AGAGGAGCCT GAGGACCTCA 1001 GGGGCCACTT GGAATCTGAC ATGTTCTCTT CACCCCTGGA GACCGACAGC ATGGACCCAT 1061 TTGGGCTTGT CACCGGACTG GAGGCCGTGA GGAGTCCTAG CTTTGAGAAG TAA Pan troglodytes gene coding sequence (1-216) (chimpanzee) FGF19(SEQ ID NO: 286) (Ensembl Accession No. ENSPTRT00000007454, which is hereby incorporated by reference in its entirety)    1 ATGCGGAACG GGTGTGTGGT GGTCCACGTC TGGATCCTGG CCGGCCTCTG GCTGGCCGTG   61 GCCGGGCGCC CCCTCGCCTT CTCGGACGCG GGGCGCCACG TGCACTACTG CTGGGGCGAC  121 CCCATCCCCC TGCGGCACCT GTACACCTCC GGCCCCCATG GGCTCTCCAG CTGCTTCCTG  181 CGCATCCCTG CGAACTGCGT CATGAACTGC GCGCGGGGCC AGAGCGCGCA CAGTTTGCTG  241 GAGATCAAGG CAGTCGCTCT GCGGACCGTG GCCATCAAGG GCGTGCACAG CGTGCGGTAC  301 CTCTGCATGG GCGCCGACGG CAAGATGCAG GGGCTGCTTC AGTACTCGGA GGAAGACTGT  361 GCTTTCGAGG AGGAGATCCG CCCAGATGGC TACAATGTGT ACCGATCCGA GAAGCACCGC  421 CTCCCGGTCT CCCTGAGCAG TGCCAAACAG CGGCAGCTGT ACAAGAACAG AGGCTTTCTT  481 CCACTCTCTC ATTTCCTGCC CATGCTGCCC ATGGTCCCAG AGGAGCCTGA GGACCTCAGG  541 GGCCACTTGG AATCTGACAT GTTCTCTTCG CCCCTGGAGA CCGACAGCAT GGACCCATTT  601 GGGCTTGTCA CCGGACTGGA GGCCGTGAGG AGTCCCAGCT TTGAGAAGTA A Macaca mulatta gene coding sequence (1-216) (Rhesus monkey) FGF19 (SEQ ID NO: 287) (GenBank Accession No. XM_001100825, which is hereby incorporated by reference in its entirety)  758 ATG AGGAGCGGGT GTGTGGTGGT CCACGCCTGG ATCCTGGCCA GCCTCTGGCT  811 GGCCGTGGCC GGGCGTCCCC TCGCCTTCTC GGACGCGGGG CCCCACGTGC ACTACGGCTG  871 GGGCGACCCC ATCCGCCTGC GGCACCTGTA CACCTCCGGC CCCCATGGGC TCTCCAGCTG  931 CTTCCTGCGC ATCCGCACCG ACGGCGTCGT GGACTGCGCG CGGGGCCAAA GCGCGCACAG  991 TTTGCTGGAG ATCAAGGCAG TAGCTCTGCG GACCGTGGCC ATCAAGGGCG TGCACAGCGT 1051 GCGGTACCTC TGCATGGGCG CCGACGGCAA GATGCAGGGG CTGCTTCAGT ACTCAGAGGA 1111 AGACTGTGCT TTCGAGGAGG AGATCCGCCC TGATGGCTAC AATGTATACC GATCCGAGAA 1171 GCACCGCCTC CCGGTCTCTC TGAGCAGTGC CAAACAGAGG CAGCTGTACA AGAACAGAGG 1231 CTTTCTTCCG CTCTCTCATT TCCTACCCAT GCTGCCCATG GCCCCAGAGG AGCCTGAGGA 1291 CCTCAGGGGC CACTTGGAAT CTGACATGTT CTCTTCGCCC CTGGAGACTG ACAGCATGGA 1351 CCCATTTGGG CTTGTCACCG GACTGGAGGC GGTGAGGAGT CCCAGCTTTG AGAAATAA Pongo abelii gene coding sequence (1-216) (Sumatran orangutan) FGF19 (SEQ ID NO: 288) (GenBank Accession No. XM_002821413, which is hereby incorporated by reference in its entirety)  763   ATGCGGAG CGGGTGTGTG GTGGTCCACG CCTGGATCCT GGCCGGCCTC TGGCTGGCCG  821 TGGCCGGGCG CCCCCTCGCC TTCTCGGACT CGGGGCCCCA CGTGCACTAC GGCTGGGGCG  881 ACCCCATCCG CCTGCGGCAC CTGTACACCT CCGGCCCCCA CGGGCTCTCC AGCTGCTTCC  941 TGCGCATCCG TGCCGACGGC GTCGTGGACT GCGCGCGGGG CCAGAGCGCG CACAGTTTGC 1001 TGGAGATCAA GGCAGTCGCT CTGCGGACCG TGGCCATCAA GGGCGTGCAC AGCGTGCGGT 1061 ACCTCTGCAT GGGCGCCGAC GGCAAGATGC AGGGGCTGCT TCAGTACTCG GAGGAAGACT 1121 GTGCTTTCGA GGAGGAGATC CGCCCAGATG GCTACAATGT GTACCGATCC GAGAAGCACC 1181 GCCTCCCGGT CTCCCTGAGC AGTGCCAAAC AGCGGCAGCT GTACAAGAAC AGGGGCTTTC 1241 TTCCGCTCTC TCATTTCCTG CCCATGCTGC CCATGGTCCC AGAGGAGCCT GAGGACCTCA 1301 GGCGCCACTT GGAATCCGAC ATGTTCTCTT CGCCCCTGGA GACCGACAGC ATGGACCCAT 1361 TTGGGCTTGT CACCGGACTG GAGGCCGTGA GGAGTCCCAG CTTTGAGAAA TAA Nomascus leucogenys gene coding sequence (1-216) (Northern white- cheeked gibbon) FGF19 (SEQ ID NO: 289) (Genbank Accession No. XM_003278023, which is hereby incorporated by reference in its entirety)  456      ATGCG GAGCGAGTGT GTGGTGGTCC ACGCCTGGAT CCTGGCCGGC CTCTGGCTGG  511 CAGTGGCCGG GCGCCCCCTC GCCTTTTCGG ACGCGGGGCC CCACGTGCAC TACGGCTGGG  571 GCGACCCCAT CCGTCTGCGG CACCTGTACA CCTCCGGCCC CCACGGGCTC TCCAGCTGCT  631 TCCTGCGCAT CCGTGCCGAC GGCGTCGTGG ACTGCGCGCG GGGCCAGAGC GCGCACAGTT  691 TGCTGGAGAT CAAGGCAGTC GCTCTGCGGA CCGTGGCCAT AAAGGGCGTG CACAGCGTGC  751 GGTACCTCTG CATGGGCGCC GACGGCAAGA TGCAGGGGCT GCTTCAGTAT TCGGAGGAAG  811 ACTGTGCTTT CGAGGAGGAG ATCCGCCCAG ATGGCTACAA TGTGTACCGA TCCGAGAAGC  871 ACCGCCTCCC CGTCTCCCTG AGCAGTGCCA AACAGCGGCA GCTGTATAAG AACAGAGGCT  931 TTCTTCCACT CTCTCATTTC CTGCCCATGC TGCCCATGGT CCCAGAGGAG CCTGAGGACC  991 TCAGGGGCCA CTTGGAATCT GACATGTTCT CTTCGCCCCT GGAGACCGAC AGCATGGACC 1051 CATTTGGGCT TGTCACCGGA CTGGAGGCCG TGAGGAGTCC CAGCTTTGAG AAATAA Callithrix jacchus gene coding sequence (1-142) (white-tufted-ear marmoset) FGF19 (SEQ ID NO: 290) (GenBank Accession No. XM_002763684, which is hereby incorporated by reference in its entirety)    1 ATGTGGAAGG CCACCGCTGG TGGCCAGCAG GGACAGTCCG AAGCACAAAT GTCCACATGT   61 CCCCATGTTC CTCGTCCTCT GTGGATTGCT CAGAGCTGCC TGTTTTCTCT GCAGCTCCAG  121 TACTCGGAGG AAGACTGTGC TTTCGAGGAG GAGATCCGCC CTGATGGCTA CAATGTGTAC  181 TGGTCCGAGA AGCACCGCCT CCCGGTCTCC CTGAGCAGCG CCAAACAGCG GCAGCTGTAC  241 AAGAAACGAG GCTTTCTTCC ACTGTCCCAT TTCCTGCCCA TGCTGCCCAT AGCCCCAGAA  301 GAGCCTGAGG ACCTCAGGGG ACACCTGGAA TCTGACGTGT TCTCTTCACC CCTGGAGACT  361 GACAGCATGG ACCCATTTGG GCTTGTCACG GGACTGGAGG CGGTGAACAG TCCCAGCTTT  421 GAGAAGTAA Microcebus murinus gene coding sequence (1-219)(mouse lemur) FGF19 (SEQ ID NO: 291) (Ensembl Accession No. ENSMICT00000003065, which is hereby incorporated by reference in its entirety)    1 ATGCCGAGCG GGCAAAGCGG TTGTGTGGCG GCCCGCGCCC TGATCCTGGC CGGCCTCTGG   61 CTGACCGCGG CCGGGCGCCC GCTGGCCTTC TCCGACGCGG GCCCGCACGT GCACTACGGC  121 TGGGGCGAGC CCATCCGCCT GCGGCACCTG TACACCGCCG GCCCCCACGG CCTCTCCAGC  181 TGCTTCCTGC GCATCCGCGC AGACGGCTCC GTGGACTGCG CGCGGGGCCA GAGCGCACAC  241 AGTTTGCTGG AGATCAGGGC GGTCGCTCTT CGGACTGTGG CCATCAAGGG CGTGCACAGC  301 GTGCGGTACC TCTGCATGGG CGCAGACGGC AGGATGCAGG GGCTGCTCCG GTACTCGGAG  361 GAAGACTGTG CCTTCGAGGA GGAGATCCGC CCCGATGGCT ACAACGTGTA CCGGTCTGAG  421 AAGCACCGCC TGCCGGTGTC TCTGAGCAGC GCCAGGCAGA GGCAGCTGTA CAAGGGCAGG  481 GGCTTCCTGC CGCTCTCTCA CTTCCTGCCC ATGCTGCCCG TGACCCCGGC AGAGACCGGG  541 GACCTCAGGG ACCACTTGGA GTCCGACATG TTCGCTTCGC CCCTGGAGAC CGACAGCATG  601 GACCCGTTTG GGATCGCCAC CAGACTTGGG GTGGTGAAGA GTCCCAGCTT TCAGAAATGA Choloepus hoffmanni gene coding sequence (1-138) (sloth) FGF19 (SEQ ID NO: 292) (Ensembl Accession No. ENSCHOT00000002324, which is hereby incorporated by reference in its entirety)    1 TTGCTCGAAA TGAAGGCAGT GGCGCTGCGG GCCGTGGCCA TCAAGGGCGT GCACAGTGCT   61 CTGTACCTCT GCATGAACGC CGACGGCAGT CTGCACGGGC TGCCTCGGTA CTCTGCAGAA  121 GACTGTGCTT TTGAGGAGGA AATCCGCCCC GACGGCTACA ATGTGTACTG GTCTAGGAAG  181 CACGGCCTCC CTGTCTCTTT GAGCAGTGCA AAACAGAGGC AGCTGTACAA AGGCAGAGGC  241 TTTCTGCCCC TGTCCCACTT CCTGCCCATG CTGCCCATGA CGCCGGCCGA GCCCGCAGAC  301 CCCGGGGATG ACGTGGAGTC GGACATGTTC TCTTCACCTC TGGAAACCGA CAGCATGGAT  361 CCTTTTGGAA TTGCCTCCAG ACTTGAGCTT GTGAACAGTC CAGCTTTCAG CATAA Ailuropoda melanoleuca gene coding sequence (124-328) (giant panda) FGF19 (SEQ ID NO: 293) (GenBank Accession No. XM_002927906, which is hereby incorporated by reference in its entirety)   69         GG TCCTAGCCGG CCTCTGCCTG GCGGTAGCCG GGCGCCCCCT AGCCTTCTCG  421 GACGCGGGGC CGCACGTGCA CTACGGCTGG GGTGAGCCCA TCCGCCTACG GCACCTGTAC  481 ACCGCCGGCC CCCACGGCCT CTCCAGCTGC TTCCTGCGCA TCCGTGCCGA CGGCGGGGTT  541 GACTGCGCGC GGGGCCAGAG CGCGCACAGT TTGGTGGAGA TCAGGGCAGT CGCTCTGCGG  601 ACCGTGGCCA TCAAGGGTGT GCACAGCGTC CGGTACCTCT GCATGGGCGC GGACGGCAGG  661 ATGCAAGGGC TGCCTCAGTA CTCTGCAGGG GACTGTGCTT TCGAGGAGGA GATCCGCCCC  721 GACGGCTACA ATGTGTACCG GTCCAAGAAG CACCGTCTCC CCGTCTCTCT GAGCGGTGCC  781 AAACAGAGGC AGCTTTACAA AGACAGAGGC TTTCTGCCCC TGTCCCACTT CTTGCCCATG  841 CTGCCCGGGA GCCCAGCAGA GCCCAGGGAC CTCCAGGACC ATGCGGAGTC GGACGGGTTT  901 TCTGCACCCC TAGAAACAGA CAGCATGGAC CCTTTTGGGA TCGCCACCAA AATGGGACTA  961 GTGAAGAGTC CCAGCTTCCA GAAATAA Sus scrofa gene coding sequence (1-218) (pig) FGF19 (SEQ ID NO: 294) (Ensembl Accession No. ENSSSCT00000014068, which is hereby incorporated by reference in its entirety)    1 ATGCGGAGCG CTCCGAGCCG GTGCGCGGTG GTCCGCGCCC TGGTCCTGGC CGGCCTCTGG   61 CTGGCCGCAG CCGGGCGCCC CCTAGCCTTC TCGGATGCTG GGCCGCACGT GCACTACGGC  121 TGGGGCGAGT CGGTCCGCCT GCGGCACCTG TACACTGCGA GTCCCCACGG CGTCTCCAGC  181 TGCTTCCTGC GCATCCACTC AGACGGCCCC GTGGACTGCG CGCCGGGACA GAGCGCGCAC  241 AGTTTGATGG AGATCAGGGC AGTCGCGCTG AGTACCGTGG CGATCAAGGG CGAGCGCAGC  301 GGCCGTTACC TCTGCATGGG CGCCGACGGC AAGATGCAAG GGCAGACTCA GTACTCGGAT  361 GAGGACTGTG CTTTCGAGGA GGAGATCCGC CCTGATGGCT ACAACGTGTA CTGGTCCAAG  421 AAACACCATC TGCCCGTCTC TCTGAGCAGC GCCAGGCAGA GGCAGCTGTA CAAAGGCAGG  481 GGCTTCCTGC CGCTGTCCCA CTTTCTGCCC ATGCTGTCCA CTCTCCCAGC CGAGCCGGAG  541 GACCTCCAGG ACCCCTTCAA GTCCGACCTG TTTTCTTTGC CCCTGGAAAC GGACAGCATG  601 GACCCTTTCC GGATCGCCGC CAAACTGGGA GCGGTGAAGA GTCCCAGCTT CTATAAATAA Bos taurus gene coding sequence (136-353)(bovine) FGF19 (SEQ ID NO: 295) (GenBank Accession No. XM_599739, which is hereby incorporated by reference in its entirety)  406                                                  ATGCG GAGCGCTCCG  421 AGCCGGTGCG CCGTGGCCCG CGCCCTGGTC CTGGCTGGCC TCTGGCTGGC CGCAGCCGGG  481 CGCCCCCTGG CCTTCTCGGA TGCGGGGCCG CACGTGCACT ACGGCTGGGG CGAGTCGGTT  541 CGCTTGCGGC ACCTGTATAC CGCGGGCCCG CAGGGCCTCT ACAGCTGCTT TCTGCGCATC  601 CACTCCGACG GCGCCGTGGA CTGCGCGCAG GTCCAGAGCG CGCACAGTTT GATGGAGATC  661 AGGGCGGTCG CTCTGAGCAC CGTAGCCATC AAGGGCGAGC GCAGCGTGCT GTACCTCTGC  721 ATGGACGCCG ACGGCAAGAT GCAAGGACTG ACCCAGTACT CAGCCGAGGA CTGTGCTTTC  781 GAGGAGGAGA TCCGTCCTGA CGGCTACAAC GTGTACTGGT CCAGGAAGCA CCATCTCCCG  841 GTCTCCCTGA GCAGCTCCAG GCAGAGGCAG CTGTTCAAAA GCAGGGGCTT CCTGCCGCTG  901 TCTCACTTCC TGCCCATGCT GTCCACCATC CCAGCCGAAC CTGAAGACCT CCAGGAACCC  961 CTGAAGCCTG ATTTCTTTCT GCCCCTGAAA ACAGATAGCA TGGACCCTTT CGGGCTCGCC 1021 ACCAAACTGG GATCGGTGAA GAGTCCCAGC TTCTATAATT AA Canis lupus familiaris gene coding sequence (1-192)(dog) FGF19 (SEQ ID NO: 296) (GenBank Accession No. XM_540802, which is hereby incorporated by reference in its entirety)    1 CTAGCCTTCT CCGACGCGGG GCCGCACGTG CACTCCTTCT GGGGGGAGCC CATCCGCCTG   61 CGGCACCTGT ACACCGCCGG CCCCCACGGC CTCTCCAGCT GCTTCCTGCG CATCCGCGCC  121 GACGGCGGGG TGGACTGCGC GCGGGGCCAG AGCGCGCACA GTCTGATGGA GATGAGGGCG  181 GTCGCTCTGC GGACCGTGGC CATCAAGGGC GTGCACAGCG GCCGGTACCT CTGCATGGGC  241 GCCGACGGCA GGATGCAAGG GCTGCCTCAG TACTCCGCCG GAGACTGTAC TTTCGAGGAG  301 GAGATCCGTC CCGATGGCTA CAATGTGTAC TGGTCCAAGA AGCACCATCT CCCCATCTCT  361 CTGAGTAGTG CCAAACAGAG GCAGCTCTAC AAGGGCAGGG GCTTTTTGCC CCTGTCCCAC  421 TTCTTACCTA TCTTGCCCGG GAGCCCAACA GAGCCCAGGG ACCTGGAAGA CCATGTGGAG  481 TCTGACGGGT TTTCTGCATC CCTGGAAACA GACAGCATGG ACCCTTTTGG GATCGCCACC  541 AAAATTGGAC TAGTGAAGAG TCCCAGTTTC CAAAAATAA Oryctolagus cuniculus gene coding sequence (1-218) (rabbit) FGF19 (SEQ ID NO: 297) (GenBank Accession No. XM_002724449, which is hereby incorporated by reference in its entirety)    1 ATGCGCCGCG CGCCGAGCGG AGGTGCCGCG GCCCGCGCCT TGGTCCTGGC CGGCCTCTGG   61 CTGGCCGCGG CCGCGCGCCC CTTGGCCTTG TCCGACGCGG GCCCGCATCT GCACTACGGC  121 TGGGGCGAGC CCGTCCGCCT GCGGCACCTG TACGCCACCA GCGCCCACGG CGTCTCGCAC  181 TGCTTCCTGC GTATACGCGC CGACGGCGCC GTGGACTGCG AGCGGAGCCA GAGCGCACAC  241 AGCTTGCTGG AGATCCGAGC GGTCGCCCTG CGCACCGTGG CCTTCAAGGG CGTGCACAGC  301 TCCCGCTACC TCTGCATGGG CGCCGACGGC AGGATGCGGG GGCAGCTGCA GTACTCGGAG  361 GAGGACTGTG CCTTCCAGGA GGAGATCAGC TCCGGCTACA ACGTGTACCG CTCCACGACG  421 CACCACCTGC CCGTGTCTCT GAGCAGTGCC AAGCAGAGAC ACCTGTACAA GACCAGAGGC  481 TTCCTGCCCC TCTCCCACTT CCTGCCCGTG CTGCCCCTGG CCTCCGAGGA GACCGCGGCC  541 CTCGGCGACC ACCCTGAAGC CGACCTGTTC TCCCCGCCCC TGGAAACCGA CAGCATGGAC  601 CCCTTCGGCA TGGCCACCAA GCTCGGGCCG GTGAAGAGCC CCAGCTTTCA GAAGTAG Pteropus vampyrus gene coding sequence (1-216) (megabat) FGF19 (SEQ ID NO: 298) (Ensembl Accession No. ENSPVAT00000009907, which is hereby incorporated by reference in its entirety)    1 ATGCGGAGCC CGTGCGCTGT GGCCCGCGCC TTGGTCCTGG CCGGCCTCTG GCTGGCCTCA   61 GCTGCGGGCC CCCTCGCCCT CTCGGACGCG GGGCCGCACG TGCACTACGG CTGGGGCGAG  121 GCCATCCGCC TGCGGCACCT GTACACCGCC GGCCCCCACG GCCCCTCCAG CTGCTTCCTG  181 CGCATCCGCG CGGATGGGGC GGTGGACTGC GCGCGGGGCC AGAGCGCGCA CAGTTTGGTG  241 GAAATCCGGG CTGTCGCCCT GCGGAACGTG GCTATCAAGG GCGTGCACAG CGTCCGATAC  301 CTCTGCATGG GAGCCGACGG CAGGATGCTA GGGCTGCTTC AGTACTCCGC TGACGACTGC  361 GCCTTCGAGG AGGAGATCCG CCCGGACGGC TACAACGTGT ACCACTCCAA GAAGCACCAC  421 CTCCCGGTCT CTCTGAGCAG TGCCAAGCAG AGGCAACTGT ACAAGGACAG GGGCTTCCTG  481 CCCCTGTCCC ATTTCCTGCC CATGCTGCCC AGGAGCCCGA CAGAGCCCGA GAACTTCGAA  541 GACCACTTGG AGGCCGACAC GTTTTCCTCG CCCCTGGAGA CAGACGACAT GGACCCTTTT  601 GGGATTGCCA GTAAATTGGG GCTGGAGGAA AGTCCCAGCT TCCAGAAGTA A Tursiops truncatus gene coding sequence (1-219) (dolphin) FGF19 (SEQ ID NO: 299) (Ensembl Accession No. ENSTTRT00000000066, which is hereby incorporated by reference in its entirety)    1 ATGCGGAGCG CTCCGAGCCG GTGCGCCGTG GCCCGCGCCC TGGTCCTGGC CGGCCTCTGG   61 CTGGCTGCAG CCGGGCGCCC CCTAGCCTTC TCGGATGCCG GGCCGCACGT GCACTACGGC  121 TGGGGCGAGT CCGTCCGCCT GCGGCACCTG TACACCGCGG GTCCCCAGGG CCTCTCCAGC  181 TGCTTCCTGC GCATCCACTC AGACGGCGCC GTGGACTGCG CGCCGGTTCA GAGCGCGCAC  241 AGTTTGATGG AGATCAGGGC AGTCGCTCTG AGTACCGTGG CCATCAAGGG CGAACGCAGC  301 GTCCTGTACC TCTGCATGGG CGCCGACGGC AAAATGCAAG GGCTGAGTCA GTACTCAGCT  361 GAGGACTGTG CCTTTGAGGA GGAAATCCGT CCGGACGGCT ACAACGTGTA CTGGTCCAAG  421 AAACACCACC TCCCGGTGTC CCTGAGCAGC GCCAGGCAGC GGCAGCTGTT CAAAGGCAGG  481 GGTTTCCTGC CGCTGTCTCA CTTCCTTCCC ATGCTGTCCA CCATCCCCAC AGAGCCCGAT  541 GAAATCCAGG ACCACTTGAA GCCCGATTTG TTTGCTTTGC CCCTGAAAAC AGATAGCATG  601 GACCCATTTG GGCTCGCCAC CAAACTGGGA GTGGTGAAGA GTCCCAGCTT CTATAAGTAA Myotis lucifugus gene coding sequence (1-219) (microbat) FGF19 (SEQ ID NO: 300) (Ensembl Accession No. ENSMLUT00000002508, which is hereby incorporated by reference in its entirety)    1 ATGCAAAGCG CGTGGAGCCG ACGCGTTGTG GCCCGAGCCC TGGTCTTGGC CAGCCTCGGG   61 CTGGCCTCAG CCGGGGGGCC CCTCGGTCTT TCGGACGCTG GGCCGCACGT GCACTACGGC  121 TGGGGGGAGT CCATCCGCCT GCGCCACCTG TACACCTCCG GCCCCCACGG CCCATCCAGC  181 TGCTTCCTGC GCATCCGCGC TGACGGCGCA GTGGACTGCG CGCGGGGCCA GAGCGCGCAC  241 AGTTTGGTGG AGATCAGGGC CGTCGCCTTG CGGAAAGTGG CCATCAAGGG CGTGCACAGC  301 GCCCTGTACC TCTGCATGGG AGGCGACGGC AGGATGCTGG GGCTGCCTCA GTTCTCGCCC  361 GAGGACTGTG CTTTCGAGGA GGAGATCCGC CCGGACGGCT ACAACGTGTA CCGGTCCCAG  421 AAGCACCAGC TGCCCGTCTC GCTGAGCAGT GCCCGGCAGA GGCAGCTGTT CAAGGCCCGG  481 GGCTTCCTGC CGCTGTCCCA CTTCCTGCCC ATGCTGCCCA GCAGCCCCGC GGGACCCGTG  541 CCCCGAGAGC GCCCCTCGGA GCCGGACGAG TTCTCTTCGC CCCTGGAAAC AGACAGCATG  601 GACCCTTTTG GGATTGCCAA CAACCTGAGG CTGGTGAGAA GTCCCAGCTT TCAGGAATAA Ornithorhynchus anatinus gene coding sequence (1-185) (platypus) FGF19 (SEQ ID NO: 301) (GenBank Accession No. XM_001506664, which is hereby incorporated by reference in its entirety)    1 ATGCTTTCCT GTGTGGTTTT GCCTAGTCTG CTGGAGATCA AGGCGGTGGC CGTGCGCACG   61 GTGGCCATCA AAGGGGTCCA CATCTCTCGG TACCTCTGCA TGGAAGAGGA TGGGAAAACT  121 CCATGGGCAC GTCTGCTGGA GATCAAGGCG GTGGCCGTGC GCACGGTGGC CATCAAAGGG  181 GTCCACAGCT CTCGGTACCT CTGCATGGAA GAGGATGGAA AACTCCATGG GCAGATTTGG  241 TATTCTGCAG AAGACTGTGC TTTTGAAGAG GAAATACGTC CAGATGGCTA CAATGTGTAT  301 AAATCTAAGA AATATGGTGT TCCTGTTTCT TTAAGCAGCG CCAAACAAAG GCAGCAATTC  361 AAAGGAAGAG ACTTTCTGCC TCTTTCTCGT TTCTTGCCAA TGATCAACAC AGTGCCTGTG  421 GAGCCAGCAG AGTTTGGGGA CTATGCCGAT TACTTTGAAT CAGATATATT TTCCTCACCT  481 CTGGAAACTG ACAGCATGGA CCCATTTAGA ATTGCCCCTA AACTGTCCCC TGTAAAGAGC  541 CCCAGCTTTC AGAAATAA Monodelphis domestica gene coding sequence (1-212) (opossum) FGF19 (SEQ ID NO: 302) (GenBank Accession No. XM_001373653, which is hereby incorporated by reference in its entirety)    1 ATGGCCCAGC TCCTGGCCCC GCTCCTCACC CTGGCTGCTC TCTGGCTGGC CCCGACGGCG   61 CGTGCCCGAC CGCTGGTGGA CGCCGGGCCT CACGTCTACT ACGGCTGGGG GGAGCCCATT  121 CGTCTGCGGC ATCTCTACAC GGCCAATCGG CACGGGCTCG CCAGCTTCTC CTTCCTCCGG  181 ATCCACCGCG ACGGCCGCGT GGACGGCAGC CGGAGTCAGA GCGCGCTCAG TTTGCTGGAG  241 ATCAAGGCGG TAGCTCTTCG GATGGTGGCG ATCAAAGGTG TCCATAGCTC TCGGTACCTG  301 TGTATGGGAG ACGCCGGGAA ACTCCAGGGA TCGGTGAGGT TCTCGGCCGA GGACTGCACC  361 TTCGAGGAGC AGATTCGCCC CGACGGCTAC AACGTGTACC AGTCCCCCAA GTACAACCTC  421 CCCGTCTCGC TCTGCACTGA CAAGCAGAGG CAGCAGGCCC ACGGCAAGGA GCACCTGCCC  481 CTGTCCCACT TCCTGCCCAT GATCAATGCT ATTCCTTTGG AGGCCGAGGA GCCCGAGGGC  541 CCCAGGATGT TGGCGGCGCC TCTGGAGACG GACAGCATGG ACCCCTTCGG CCTCACCTCC  601 AAGCTGTTGC CGGTCAAGAG CCCCAGCTTT CAGAAATAA Anolis carolinensis gene coding sequence (1-220) (anole lizard) FGF19 (SEQ ID NO: 303) (GenBank Accession No. XM_003214667, which is hereby incorporated by reference in its entirety)    1 ATGTGTCGGC GGGCGTTGCC TCTGCTGGGG GCCCTTCTGG GCTTGGCGGC CGTGGCCTCC   61 CGCGCCCTCC CGCTCACCGA CGCCGGGCCC CACGTCAGCT ACGGCTGGGG GGAGCCCGTC  121 CGGCTCAGGC ACCTCTACAC CGCGGGGCGG CAGGGCCTCT TCAGCCAGTT CCTCCGCATC  181 CACGCCGACG GGAGAGTCGA CGGCGCCGGC AGCCAGAACC GGCAGAGTTT GCTGGAGATC  241 CGCGCGGTCT CGTTGCGCGC CGTGGCCCTC AAAGGCGTGC ACAGCTCCCG CTACCTCTGC  301 ATGGAGGAGG ACGGCCGGCT CCGCGGGATG CTCAGATATT CTGCAGAAGA CTGTTCCTTT  361 GAAGAGGAGA TGCGTCCAGA TGGCTACAAT ATCTACAAGT CAAAGAAATA CGGAGTTTTG  421 GTCTCCCTAA GTAATGCCAG ACAAAGACAG CAATTCAAAG GGAAAGATTT TCTTCCTTTG  481 TCTCATTTCT TGCCGATGAT CAACACTGTG CCAGTGGAGT CTGCAGACTT TGGAGAGTAT  541 GGTGACACCA GGCAGCATTA TGAATCGGAT ATTTTCAGTT CACGTCTTGA AACTGACAGC  601 ATGGACCCTT TTGGCCTCAC TTCAGAAGTG TCATCAGTAC AAAGTCCTAG CTTTGGGAAA  661 TAA Ochotona princeps gene coding sequence (1-214, excluding 78-112)(pika) FGF19 (SEQ ID NO: 304) (Ensembl Accession No. ENSOPRT00000010769, which is hereby incorporated by reference in its entirety)    1 GTGCGGAGCA GGGGAGCCAT GGCCCGCGCT CTGGTTCTAG CCACTCTCTG GCTGGCCGCG   61 ACGGGGCGGC CGCTGGCCTT GTCCGACGCG GGGCCGCACC TGCACTACGG CTGGGGCGAG  121 CCCATCCGCC TGCGGCACCT GTACGCCACC AGCGCCCACG GCCTCTCGCA CTGCTTTTTG  181 CGCATCCGTA CCGACGGCAC CGTGGACTGC GAGCGCAGCC AGAGCGCGCA CA--------     ---------- ---------- ---------- ---------- ---------- ---------- 242 ---------- ---------- ---------- ------CTAC AGTACTCGGA GGAGGACTGC  266 GCCTTCGAAG AGGAGATCAG CTCTGGCTAT AACGTGTACC GCTCCAGGAG GTACCAGCTG  326 CCCGTGTCCC TGGGCAGCGC CAGGCAGAGG CAGCTGCAGC GGAGCCGTGG CTTCCTGCCC  386 CTGTCCCACT TCCTGCCGGT GCTGCCCGCG GCCTCGGAGG AGGTGGCGGC CCCCGCTGAC  446 CACCCGCAAG CAGACCCTTT CTCGCCCCTG GAGACCGACA GCATGGACCC ATTTGGAATG  506 GCCACCAAGC GGGGGCTGGT GAAGAGCCCC AGCTTCCAGA AGTGA Cavia porcellus gene coding sequence (1-221)(guinea pig) FGF19 (SEQ ID NO: 305) (Ensembl Accession No. ENSCPOT00000008222, which is hereby incorporated by reference in its entirety)    1 ATGTGGAGTG CGCCGAGCGG ATGTGTGGTG ATCCGCGCCC TGGTCCTGGC TGGCCTGTGG   61 CTGGCGGTGG CGGGGCGCCC CCTGGCCCGG CGGTCTCTCG CGCTATCTGA CCAGGGGCCG  121 CACTTGTACT ACGGCTGGGA CCAGCCGATC CGCCTTCGGC ACCTGTACGC CGCGGGCCCC  181 TACGGCCGCT CGCGCTGCTT CCTGCGCATT CACACGGACG GCGCGGTGGA CTGCGTCGAG  241 GAACAGAGCG AGCACTGTTT GCTGGAGATC AGAGCAGTCG CTCTGGAGAC CGTGGCCATC  301 AAGGACATAA ACAGCGTCCG GTACCTGTGC ATGGGCCCCG ACGGCAGGAT GCGGGGCCTG  361 CCCTGGTATT CGGAGGAGGA CTGTGCCTTC AAGGAAGAGA TCAGCTACCC GGGCTACAGC  421 GTGTACCGCT CCCAGAAGCA CCACCTCCCC ATCGTGCTGA GCAGTGTCAA GCAGAGGCAG  481 CAGTACCAGA GCAAGGGGGT GGTGCCCCTG TCCTACTTCC TGCCCATGCT GCCCAAGGCC  541 TCTGTGGAGC CCAGCGACGA GGAGGAATCC AGCGTGTTCT CGTTGCCCCT GAAGACGGAC  601 AGCATGGACC CCTTTGGGAT GGCCAGTGAG ATCGGGCTGG TGAAGAGTCC CAGCTTTCAG  661 AAGTAA Tupaia belangeri gene coding sequence (1-219, excluding 116-138)(tree shrew) FGF19 (SEQ ID NO: 306) (from Ensembl Accession No. ENSTBET00000000307, which is hereby incorporated by reference in its entirety)   1 ATGAGGAGAA CACCGAGCGG GTTTGCAGTG GCCCGTGTCC TCTTCCTGGG CAGCCTTTGG   61 CTGGCCGCAG CCGGGAGCCC CTTGGCCCTG TCCGACGCCG GGCCGCATGT GAACTACGGC  121 TGGGATGAGT CCATACGCCT GCGACACTTG TACACCGCCA GCCCGCACGG CTCCACCAGC  181 TGCTTCTTGC GCATCCGTGA CGACGGCTCA GTGGACTGCG CGCGGGGCCA GAGTTTGCAC  241 AGTTTGCTGG AGATCAAGGC AGTCGCTTTG CAGACCGTGG CCATCAAAGG CGTGTACAGT  301 GTCCGCTACC TCTGCATGGA CGCCGACGGC AGGATGCAGG GGCTG----- ---------- 361 ---------- ---------- ---------- ---------- ---------- NNGGTCCACG  369 AAGCACGGCC TCCCAGTCTC CCTGAGCAGT GCCAAGCAGA GGCAGCTGTT AACGGTTAGG  429 GGCTTTCCTT CCCTTCCCCA CTTCCTGCTC ATGATGGCCA AGACTTCAGC AGGGCCTGGA  489 AACCCCAGGG ACCACCCAGG GTCTAACACT TTCTCGTTGC CCCTGGAAAC TGATAGCATG  549 GACCCATTTG GGATGACCAC CAGACATGGG CTGGTGAAGA GTCCCAGCTT TCAAAACTAA Rattus norvegicus gene coding sequence (1-218)(Norway rat) FGF15(GenBank Accession No. NM_130753, which is hereby incorporated by reference in its entirety) (SEQ ID NO: 307)    1 ATGGCGAGAA AGTGGAGTGG GCGTATTGTG GCCCGAGCTC TGGTCCTGGC CACTCTGTGG   61 CTGGCCGTGT CTGGGCGTCC CCTGGTCCAG CAATCCCAGT CTGTGTCGGA TGAAGGTCCA  121 CTCTTTCTCT ATGGCTGGGG CAAGATTACC CGCCTGCAGT ACCTGTACTC TGCTGGTCCC  181 TACGTCTCCA ACTGCTTCCT GCGTATCCGG AGTGACGGCT CTGTGGACTG CGAGGAGGAC  241 CAGAACGAAC GAAATCTGTT GGAGTTCCGC GCGGTTGCTC TGAAGACAAT TGCCATCAAG  301 GACGTCAGCA GCGTGCGGTA CCTCTGCATG AGCGCCGACG GCAAGATATA CGGGCTGATT  361 CGCTACTCGG AGGAAGACTG TACCTTCAGG GAGGAAATGG ACTGTTTGGG CTACAACCAG  421 TACAGGTCCA TGAAGCACCA CCTCCACATC ATCTTCATCA AGGCCAAGCC CAGAGAGCAG  481 CTCCAGGGCC AGAAACCTTC AAACTTTATC CCCATATTTC ACCGGTCTTT CTTTGAATCC  541 ACGGACCAGC TGAGGTCTAA AATGTTCTCT CTGCCCCTGG AGAGCGACAG CATGGATCCG  601 TTCAGAATGG TGGAGGATGT GGACCACCTA GTGAAGAGTC CCAGCTTCCA GAAATGA Mus musculus gene coding sequence (1-218)(house mouse) FGF15 (SEQ ID NO: 308) (GenBank Accession No. NM_008003, which is hereby incorporated by reference in its entirety)  148                              ATG GCGAGAAAGT GGAACGGGCG TGCGGTGGCC  181 CGAGCCCTGG TCCTGGCCAC TCTGTGGCTG GCTGTGTCTG GGCGTCCCCT GGCTCAGCAA  241 TCCCAGTCTG TGTCAGATGA AGATCCACTC TTTCTCTACG GCTGGGGCAA GATTACCCGC  301 CTGCAGTACC TGTACTCCGC TGGTCCCTAT GTCTCCAACT GCTTCCTCCG AATCCGGAGC  361 GACGGCTCTG TGGACTGCGA GGAGGACCAA AACGAACGAA ATTTGTTGGA ATTCCGCGCG  421 GTCGCTCTGA AGACGATTGC CATCAAGGAC GTCAGCAGCG TGCGGTACCT CTGCATGAGC  481 GCGGACGGCA AGATATACGG GCTGATTCGC TACTCGGAGG AAGACTGTAC CTTCAGGGAG  541 GAAATGGACT GTTTAGGCTA CAACCAGTAC AGATCCATGA AGCACCATCT CCATATCATC  601 TTCATCCAGG CCAAGCCCAG AGAACAGCTC CAGGACCAGA AACCCTCAAA CTTTATCCCC  661 GTGTTTCACC GCTCCTTCTT TGAAACCGGG GACCAGCTGA GGTCTAAAAT GTTCTCCCTG  721 CCCCTGGAGA GTGACAGCAT GGATCCGTTC AGGATGGTGG AGGATGTAGA CCACCTAGTG  781 AAGAGTCCCA GCTTCCAGAA ATGA Gallus gallus gene coding sequence (1-227)(chicken) FGF19 (SEQ ID NO: 309) (GenBank Accession No. NM_204674, which is hereby incorporated by reference in its entirety)  127       ATGG GGCCGGCCCG CCCCGCCGCA CCCGGCGCTG CCCTGGCGCT GCTGGGGATC  181 GCCGCCGCCG CCGCCGCCGC CAGGTCCCTG CCGCTGCCCG ACGTCGGGGG TCCGCACGTC  241 AACTACGGCT GGGGGGAACC CATCCGGCTG CGGCACCTAC TACACCGCCC AGGCAAGCAC  301 GGGCTCTTCA GCTGCTTCCT GCGCATCGGC GGCGACGGCC GGGTGGACGC TGTCGGTAGC  361 CAGAGCCCGC AGAGTCTGTT GGAGATCCGC GCCGTGGCGG TGCGCACCGT GGCCATCAAG  421 GGCGTGCAGA GCTCCCGCTA CCTCTGCATG GACGAGGCGG GGCGGCTGCA CGGGCAGCTC  481 AGCTATTCCA TTGAGGACTG TTCCTTTGAA GAGGAGATTC GTCCAGACGG CTACAACGTG  541 TATAAATCAA AGAAATACGG GATATCGGTG TCTTTGAGCA GTGCCAAACA AAGACAGCAA  601 TTCAAAGGAA AAGATTTTCT CCCGCTGTCT CACTTCTTAC CCATGATCAA CACTGTGCCA  661 GTGGAGGTGA CAGACTTTGG TGAATATGGT GATTACAGCC AGGCTTTTGA GCCAGAGGTC  721 TACTCATCGC CTCTCGAAAC GGACAGCATG GATCCCTTTG GGATCACTTC CAAACTGTCT  781 CCAGTGAAGA GCCCCAGCTT TCAGAAATGA Taeniopygia guttata gene coding sequence (1-237)(zebra finch) FGF19 (SEQ ID NO: 310) (GenBank Accession No. XM_002194457, which is hereby incorporated by reference in its entirety)    1 ATGGTTATCA TAAGCAATCT ATATCTGATG CAGAACGATG TTATGATGAA TATGAGGCGA   61 GCACCCCTTC GCGTTCACGC TGCTCGCTCT TCGGCCACCC CTGCCTCCGC GCTGCCGCTG  121 CCGCCGCCCG ACGCCGGCCC GCACCTCAAA TACGGCTGGG GAGAGCCCAT CCGGCTGCGG  181 CACCTCTACA CCGCCAGCAA GCACGGGCTC TTCAGCTGCT TCCTGCGTAT CGGCGCTGAC  241 GGCCGGGTGG ACGCGGCCGG CAGCCAGAGC CCGCAGAGCC TGCTAGAGAT CCGCGCCGTG  301 GCCGTGCGCA CCGTGGCCAT CAAGGGCGTG CAGAGCTCCC GGTACCTGTG CATGGACGAG  361 GCGGGGCGGC TGCACGGGCA GCTCAGGAAT TCCACTGAAG ACTGCTCCTT TGAGGAGGAG  421 ATTCGCCCAG ACGGCTACAA TGTGTATAGA TCTAAAAAAC ATGGAATATC GGTGTCTTTG  481 AGCAGTGCCA AACAAAGACA GCAGTTCAAG GGGAAAGATT TCCTTCCCCT GTCTCACTTC  541 TTGCCCATGA TCAACACTGT GCCCATGGAG TCAGCAGACT TTGGTGAATA TGGTGATTAC  601 AGCCAGGCCT TTGAGGCAGA GGCCTTCTCC TCACCTCTGG AGACGGACAG CATGGACCCC  661 TTTGGCATCG CCTCCAAACT GTCCCTAGTG AAGAGCCCTA GCTTCCAAAA CTGA Danio rerio gene coding sequence (1-210)(zebrafish) FGF19 (SEQ ID NO: 311) (GenBank Accession No. NM_001012246, which is hereby incorporated by reference in its entirety)    1 ATGCTCCTCT TACTCTTTGT CACTGTTTGT GGAAGTATCG GCGTGGAGAG CCTCCCGTTG   61 CCCGACTCTG GTCCACATTT GGCAAATGAC TGGAGTGAAG CCGTCCGGCT ACGACATCTG  121 TACGCAGCCA GACATGGCTT ACATCTGCAA ATAAACACAG ACGGAGAAAT CATTGGATCC  181 ACATGCAAAG CTCGGACAGT AAGTTTGATG GAGATATGGC CGGTGGACAC AGGCTGCGTA  241 GCCATTAAGG GAGTTGCAAG CTCCCGATTT CTTTGCATGG AAAGACTGGG AAACCTGTAC  301 GGATCGCACA TTTACACTAA AGAGGACTGC TCTTTTTTGG AACGCATCCT TCCAGACGGC  361 TACAACGTCT ACTTCTCGAG CAAACACGGA GCTCTTGTGA CTTTAAGTGG TGCGAAAAAC  421 AAGTTGCACA GTAACGATGG GACTTCTGCA TCCCAGTTCC TCCCCATGAT CAACACACTT  481 TCAGAGGAAC ACACTAAACA GCACTCAGGG GAACAGCACT CTTCTGTTAA CCATGGACAG  541 GACCATCAGT TGGGCCTTGA AATAGACAGT ATGGACCCTT TCGGAAAGAT CTCTCAAATA  601 GTGATCCAGA GTCCCAGCTT CAACAAAAGA TGA Xenopus (Silurana) tropicalis gene coding sequence (1-215)(Western clawed frog) FGF19 (SEQ ID NO: 312) (GenBank Accession No. NM_001142825, which is hereby incorporated by reference in its entirety)    1 ATGTGGAAGA CCCTGCCTTG GATTTTGGTT CCCATGATGG TGGCCGTGCT GTATTTCCTC   61 GGAGGGGCGG AAAGTCTGCC GCTTTTTGAT GCCGGGCCGC ACATGCAGAA CGGCTGGGGG  121 GAGTCGATCA GAATTCGGCA CCTGTATACG GCCAGGAGGT TCGGGCACGA CAGCTACTAC  181 CTCCGGATAC ACGAGGATGG CAGAGTCGAT GGTGACAGGC AACAAAGCAT GCACAGTTTA  241 TTGGAAATCA GAGCAATTGC AGTTGGAATT GTTGCCATTA AAGGGTATCG CAGCTCTCTG  301 TACCTGTGCA TGGGGTCCGA GGGAAAACTC TATGGAATGC ACAGTTACTC CCAGGATGAT  361 TGCTCTTTTG AAGAGGAGCT TCTCCCGGAT GGATACAACA TGTATAAATC AAGGAAACAT  421 GGCGTTGCTG TCTCCCTAAG CAAGGAGAAG CAGAAGCAAC AATACAAAGG AAAGGGCTAC  481 CTCCCGTTGT CCCATTTCCT ACCCGTGATA AGCTGGGTGC CCATGGAGCC CACCGGAGAT  541 GTAGAAGATG ATATCTACAG GTTTCCATTC AATACGGACA CAAAAAGTGT CATTGACAGC  601 CTTGATACCC TGGGACTAAT GGATTTTTCG AGTTATCACA AGAAATAG Otolemur garnettii (bushbaby) FGF19 gene coding sequence (1-219)(SEQ ID NO: 313) (Ensembl accession no. EN50GAT00000031686, which is hereby incorporated by reference in its entirety)    1 ATGCCCAGCG GGCTGAGAGG GCGTGTGGTA GCCGGCGCCC TGGCCCTGGC CAGCTTCTGG   61 CTGGCCGTGG CCGGGCGCCC GCTGGCCTTC TCGGATGCCG GCCCTCACGT GCACTACGGC  121 TGGGGTGAGC CCATCCGCCT GCGACACCTG TACACCGCCG GCCCCCACGG CCTCTCCAGC  181 TGCTTCCTGC GCGTACGCAC CGACGGTGCG GTAGACTGCG CGCGGGGCCA GAGCGCACAC  241 AGTTTGCTGG AAATCAGGGC CGTCGCTCTC CGGACCGTGG CCATCAAAGG CGTGCACAGC  301 GCGCGGTACC TCTGCATGGG CGCCGACGGC AGGATGCAGG GGCTGCCTCA GTACTCGGAG  361 GAAGACTGTG CCTTTGAGGA GGAGATCCGG CCAGACGGCT ACAACGTCTA CTGGTCTGAG  421 AAGCACCGCC TGCCGGTGTC TCTGAGCAGT GCCCGGCAGA GGCAGCTGTA CAAGGGCAGG  481 GGCTTTCTGC CGCTCTCTCA CTTCCTGCCC ATGCTGCCTG TGACCCCAGC CGAGCCCGGG  541 GACCTCAGAG ACCACCTGGA ATCCGACATG TTCTCTTTGC CCCTGGAAAC TGACAGCATG  601 GATCCATTTG GGATCGCCAC CAGACTGGGC GTGGTGAAGA GTCCCAGCTT TCAGAAATGA Felis catus (cat) FGF19 gene coding sequence (1-219)(SEQ ID NO: 314) (Ensembl accession no. ENSFCAT00000026317, which is hereby incorporated by reference in its entirety)    1 ATGCGGAGCG CGCCGAGCCA GTGCGCGGTA ACCCGCGCCC TGGTCCTAGC CGGTCTCTGG   61 CTGGCAGCAG CCGGGCGCCC CCTAGCCTTC TCGGACGCGG GGCCTCACGT GCACTACGGC  121 TGGGGTGAGC CCATCCGCCT GCGGCACCTG TACACCGCCG GCCCCCACGG CCTCTCCAGC  181 TGCTTCCTGC GCATCCGAGC CGACGGGGGG GTTGACTGCG CGCGGAGCCA GAGCGCGCAC  241 AGTTTGGTGG AGATCAGGGC AGTCGCTCTG CGGACCGTGG CCATCAAGGG CGTGCACAGC  301 GTCCGGTACC TCTGCATGGG CGCCGACGGC AGGATGCAAG GGCTGCTTCA GTACTCTGCT  361 GGGGACTGTG CCTTCCAAGA GGAGATCCGC CCCGACGGCT ACAATGTGTA CCGGTCCGAG  421 AAGCACCGTC TCCCCGTCTC TTTGAGTAGT GCCATACAGA GGCAGCTGTA CAAGGGCAGA  481 GGGTTTTTGC CCCTGTCCCA TTTCTTGCCC ATGCTGCCCG GCAGCCCAGC AGAGCCCAGG  541 GACCTCCAGG ACCACGTGGA GTCGGAGAGG TTTTCTTCAC CCCTGGAAAC AGACAGCATG  601 GACCCTTTTG GGATTGCCAC CAAAATGGGG TTAGTGAAGA GTCCCAGCTT CCAAAAGTAA Pelodiscus sinensis (Chinese softshell turtle) FGF19 gene coding sequence (1-223)(SEQ ID NO: 315) (Ensembl accession no. ENSPSIT00000010427, which is hereby incorporated by reference in its entirety)  241                                    ATGTGGAG GAGCCTGTGC AAATCTCACA  301 CGTCTCTGGC TCTGCTGGGA CTCTGCTTTG CGGTGGTCGT GAGATCTCTG CCTTTCTCGG  361 ATGCAGGGCC ACATGTGAAC TATGGCTGGG GGGAGCCTAT TCGATTAAGG CACCTATACA  421 CCGCCAGCAG ACACGGGCTG TTCAATTACT TCCTGAGGAT CAGCAGTGAT GGCAAAGTGG  481 ATGGCACCAG CATTCAGAGT CCTCACAGTC TGCTGGAAAT CAGGGCTGTG GCAGTTCGCA  541 CGGTGGCGAT CAAGGGCGTC CACAGTTCCC GGTACCTCTG CATGGAAGAA GACGGGAAGC  601 TGCATGGACT TCTCAGGTAT TCTACAGAAG ATTGCTCCTT TGAAGAGGAG ATACGCCCAG  661 ATGGCTACAA TGTATATAAA TCAAAGAAAT ATGGAATCTC TGTGTCCTTA AGTAGTGCCA  721 AACAAAGACA ACAATTCAAA GGAAAAGACT TTCTTCCATT GTCTCACTTC TTGCCTATGA  781 TCAATACAGT ACCTGTGGAG TCAATGGATT TTGGAGAATA TGGTGATTAT AGTCATACTT  841 TTGAATCAGA TCTATTCTCT TCACCTCTCG AAACTGACAG CATGGATCCC TTTGGAATCA  901 CCTCTAAAAT ATCTCCAGTG AAGAGCCCCA GCTTTCAGAA ATAA Latimeria chalumnae (coelacanth) FGF19 gene coding sequence (1- 217)(SEQ ID NO: 316) (Ensembl accession no. ENSLACT00000014697, which is hereby incorporated by reference in its entirety)     1 ATGTTACAGG CACTGTACAA TCTCTGTACA GCTCTAGTTT TGTTTAAGCT TCCTTTTGCA   61 ATGGTGGGGT ACACCCTGCC TTCTGCCAAT GAAGGGCCCC ATCTGAACTA TGACTGGGGA  121 GAATCTGTAA GACTCAAACA TCTGTACACA TCTAGCAAGC ATGGATTGAT CAGTTACTTT  181 TTACAGATCA ATGATGATGG CAAAGTAGAT GGGACCACTA CACGAAGCTG TTATAGTTTG  241 CTCGAAATAA AATCAGTGGG GCCAGGAGTT TTGGCAATTA AAGGCATACA GAGCTCCAGA  301 TACCTTTGTG TCGAGAAGGA TGGAAAATTG CATGGATCGC GCACTTATTC AGCAGACGAT  361 TGCTCCTTCA AAGAGGATAT ACTCCCAGAT GGTTACACTA TCTACGTGTC AAAGAAACAT  421 GGATCTGTTG TTAATCTGAG CAACCACAAA CAGAAACGTC AGAGAAATCG CAGAACCCTG  481 CCTCCATTTT CTCAGTTCCT ACCGCTTATG GACACCATTC GTGTGGAGTG CATGAACTGC  541 GGGGAGCACT GTGACGACAA CCTGCATGAC GAGCTAGAAA CAGGACTGTC CATGGATCCC  601 TTTGAAAGTA CATCCAAAAA ATCCTTTCAG AGTCCCAGCT TTCACAATAG ATAA Mustela putorius furo (ferret) FGF19 gene coding sequence (1-219)(SEQ ID NO: 317) (Ensembl accession no. ENSMPUT00000004650, which is hereby incorporated by reference in its entirety)  421     ATGCGG AGCGCCGCGA GTCGGTGCGC GGTAGCCCGC GCGCTGGTCC TAGCCGGCCT  481 TTGGCTGGCC GCAGCCGGGC GCCCCCTAGC CTTCTCGGAC GCGGGGCCGC ACGTGCACTA  541 TGGCTGGGGT GAGCCCATCC GCCTACGGCA CCTGTACACC GCCGGCCCCC ACGGCCTCTC  601 CAGCTGCTTC CTGCGCATCC GTGCCGACGG CGGGGTTGAC TGCGCGCGGG GCCAGAGCGC  661 GCACAGTTTG GTGGAGATCC GGGCAGTCGC TCTGCGGACG GTGGCCATCA AGGGCGTGTA  721 CAGCGACCGC TATCTCTGCA TGGGTGCGGA CGGCAGGATG CAAGGGCTGC CTCAGTACTC  781 CGCCGGAGAC TGTGCTTTCG AGGAGGAGAT CCGCCCTGAT GGCTACAACG TGTACCGGTC  841 CAAGAAGCAC CGTCTCCCCG TCTCCCTGAG CAGTGCGAAA CAAAGGCAGC TGTACAAGGA  901 CCGGGGCTTT TTGCCTCTGT CCCATTTCTT GCCCATGCTG CCCGGGAGCC TGGCGGAGCC  961 CAGGGACCTC CAGGACCACG TGGAGGCTGA TGGGTTTTCT GCCCCCCTAG AAACAGACAG 1021 CATGGACCCT TTTGGGATTG CCACCAAAAT GGGACTAGTG AAGAGTCCCA GCTTCCAAAA 1081 ATGA Takifugu rubripes (fugu) FGF19 gene coding sequence (1-218)(SEQ ID NO: 318) (Ensembl accession no. ENSTRUT00000007155, which is hereby incorporated by reference in its entirety)    1 TCATCTACAA GGATTAGTGG AAACATGGTT CTCCTCATGC TCCCCATCAC CGTTGCAAAC   61 CTCTTCCTCT GTGCTGGAGT TCTCTCCTTG CCTTTGTTGG ATCAAGGGTC TCATTTTCCC  121 CAAGGCTGGG AACAGGTAGT CCGCTTCAGG CACCTGTATG CTGCCAGTGC AGGGCTGCAC  181 CTGCTGATCA CTGAAGAGGG CTCGATCCAA GGCTCTGCAG ATCCAACTTT ATACAGCCTG  241 ATGGAGATCC GTCCGGTGGA CCCAGGCTGT GTTGTCATTA GAGGAGCAGC AACCACACGC  301 TTCCTCTGCA TAGAAGGTGC TGGAAGACTG TACTCATCAC AGACCTACAG CAAAGACGAC  361 TGTACCTTCA GAGAGCAAAT CCTAGCAGAC GGCTACAGCG TCTACAGATC TGTCGGACAC  421 GGAGCTCTGG TCAGTCTGGG AAACTACCGG CAGCAGCTGA GGGGGGAGGA CTGGAGCGTT  481 CCGACACTGG CTCAGTTCCT CCCCAGAATA AGTTCACTGG ATCAGGACTT TAAAGCTGCT  541 CTTGACGAGA CTGAGAAGCC AGAACAAACT GCACCTCAAA GATCGGAACC TGTCGACATG  601 GTGGACTCAT TTGGAAAGCT CTCTCAGATC ATCCACAGTC CCAGTTTTCA CAAG Equus caballus (horse) FGF19 gene coding sequence (1-216, excluding 1-19 and 114-216)(SEQ ID NO: 319) (Ensembl accession no. ENSECAT00000021494, which is hereby incorporated by reference in its entirety)    1 ---------- ---------- ---------- ---------- ---------- -------GCG    4 GCCGGGCGCC CCCTAGCCTT GTCCGACGCT GGGCCGCACG TGCACTACGG CTGGGGCGAG   64 CCGATCCGCC TGCGGCACCT GTACACCGCC GGCCCCCACG GCCTCTCCAG CTGCTTCCTG  124 CGCATCCGCG CCGATGGCGC CGTGGACTGC GCGCGGGGCC AGAGCGCGCA CAGTTTGGTG  184 GAGATCAGAG CAGTCGCTCT GCGCACCGTG GCCATCAAGG GCGTGCACAG CGTCCGGTAC  244 CTCTGCATGG GCGCCGACGG CAGGATGCAA GGGCTGGTA Oryzias latipes (medaka) FGF19 gene coding sequence (1-209)(SEQ ID NO: 320) (Ensembl accession no. ENSORLT00000000352, which is hereby incorporated by reference in its entirety)    1 ACCATGCTGC TCATTGTGGT CACCATTTCC ACAATGGTGT TTTCTGACTC TGGAGTTTCC   61 AGCATGCCGC TCTCTGATCA TGGACCCCAC ATCACTCACA GCTGGAGCCA AGTGGTCCGC  121 CTCCGGCACC TGTACGCGGT CAAGCCTGGA CAACATGTCC AGATCAGAGA GGATGGACAC  181 ATCCACGGCT CAGCAGAACA AACTCTGAAC AGCCTGCTGG AGATCCGTCC GGTTGCTCCG  241 GGACGGGTGG TCTTCAGAGG AGTAGCCACC TCAAGGTTTC TGTGCATGGA GAGCGACGGC  301 AGACTCTTCT CCTCACACAC ATTTGACAAG GACAACTGCG TCTTCAGAGA GCAGATCTTG  361 GCAGACGGCT ACAACATCTA CATTTCAGAT CAGCATGGAA CCCTGCTTAG TTTGGGAAAC  421 CACCGGCAAA GGCAGCAGGG TTTAGACCGG GATGTTCCAG CCCTGGCTCA GTTCCTCCCC  481 AGGATCAGCA CCCTGCAGCA GGGCGTGTAC CCAGTGCCAG ACCCCCCCCA CCAGATGAGA  541 ACAATGCAAA CAGAGAAGAC TCTAGATGCC ACGGACACAT TTGGGCAACT CTCTAAAATC  601 ATTCACAGTC CCAGCTTCAA CAAAAGATGA Xiphophorus maculatus (platyfish) FGF19 gene coding sequence (1-207) (SEQ ID NO: 321) (Ensembl accession no. ENSXMAT00000001519, which is hereby incorporated by reference in its entirety)     1 ATG    4 TTTGTGTTCA TTCTATGCAT TGCTGGTGAA CTTTTTACTC TGGGAGTATT TTGCATGCCA   64 ATGATGGACC AGGGGCCACT TGTCACCCAT GGCTGGGGCC AGGTGGTCCG GCACCGGCAT  124 CTGTATGCAG CCAAGCCAGG ACTGCACCTA CTGATCAGTG AGGATGGACA AATCCACGGT  184 TCCGCAGATC AAACTCTTTA CAGCCTGCTG GAGATCCAAC CTGTTGGCCC CGGACGTGTT  244 GTGATCAAAG GAGTGGCAAC CACACGCTTC CTCTGCATGG AGAGCGACGG CAGATTGTAC  304 TCAACTGAAA CATACAGCAG AGCTGACTGC ACCTTCAGAG AACAGATCCA GGCAGACGGC  364 TACAACGTCT ACACCTCTGA TAGCCATGGA GCCCTCCTCA GTTTGGGAAA CAACCAGCAA  424 AGACACAGCG GCTCAGACCG TGGTGTTCCA GCTCTGGCCC GCTTTCTTCC CAGGTTAAAC  484 ACCCTTCAGC AGGCCGTCCC CACAGAGCCG GATGTTCCTG ATCAGCTCAG TCCAGAGAAA  544 GTACAACAGA CTGTGGACAT GGTGGCCTCC TTTGGCAAGC TCTCTCATAT AATTCACAGT  604 CCCAGCTTCC ATAAGAGATG A Ictidomys tridecemlineatus (squirrel) FGF19 gene coding sequence (1- 220)(SEQ ID NO: 322) (Ensembl accession no. ENSSTOT00000026298, which is hereby incorporated by reference in its entirety)    1 ATGCGGAGCG CGCCGAGCGG ACGTGCCTTA GCCCGCGCCC TGGTGCTGGC CAGCCTCTGG   61 TTGGCAGTGG CCGGACGACC CCTGGCCCGG CGCTCTCTGG CTCTCTCCGA CCAGGGGCCA  121 CACTTGTACT ATGGCTGGGA TCAGCCCATC CGCCTCCGGC ACCTGTACGC CGCGGGCCCC  181 TACGGCTTCT CCAACTGTTT CCTGCGCATC CGCACCGACG GCGCCGTGGA CTGCGAGGAG  241 AAGCAGAGCG AGCGTAGTTT GATGGAGATC AGGGCGGTCG CTCTGGAGAC TGTGGCCATC  301 AAGGACATAA ACAGCGTCCG GTACCTCTGC ATGGGCGCCG ACGGCAGGAT ACAGGGACTG  361 CCTCGGTACT CGGAGGAAGA GTGCACGTTC AAGGAGGAGA TCAGCTATGA CGGCTACAAC  421 GTGTACCGGT CCCAGAAGTA CCACCTTCCC GTGGTGCTCA GCAGTGCCAA GCAGCGGCAG  481 CTGTACCAGA GCAAGGGCGT GGTTCCCCTG TCCTACTTCC TGCCCATGCT GCCCCTGGCC  541 TCTGCGGAGA CCAGGGACCG CTTGGAATCC GATGTGTTCT CTTTACCTCT GGAAACTGAC  601 AGCATGGACC CGTTTGGGAT GGCCAGTGAA GTGGGCCTGA AGAGCCCCAG CTTCCAGAAG  661 TAA Gasterosteus aculeatus (stickleback) FGF19 gene coding sequence (1- 203)(SEQ ID NO: 323) (Ensembl accession no. ENSGACT00000018770, which is hereby incorporated by reference in its entirety)    1 ATGCTGCTGC TGCTGGTCCC CGCGTACGTT GCCAGTGTGT TTTTAGCTCT CGGGGTTGTT   61 TGCTTGCCCC TAACAGATCA GGGTCTCCAC ATGGCCGACG ACTGGGGCCA GTCGGTCCGA  121 CTCAAGCACC TGTACGCCGC CAGCCCGGGA CTCCACCTGC TGATCGGGGA GGATGGTCGG  181 ATCCAAGGCT CGGCGCAGCA AAGCCCCTAC AGCCTGCTGG AGATCAGTGC AGTGGATCCG  241 GGCTGTGTGG TCATCAGAGG AGTAGCAACC GCACGGTTTC TCTGCATCGA AGGCGATGGA  301 AGACTGTACT CATCGGACAC CTACAGCAGA GACGACTGCA CCTTCAGGGA GCAGATCCTC  361 CCGGACGGCT ACAGCGTCTA CGTCTCCCAT GGACACGGGG CCCTGCTCAG CCTGGGGAAC  421 CACAGGCAGA GGCTGCAGGG TCGAGACCAC GGCGTGCCGG CTCTGGCCCA GTTCCTCCCG  481 AGGGTCAGCA CCATGGATCA GGCCTCGGCC CCCGACGCGC CCGGGCAGAC CGCCACCGAG  541 ACGGAAGAGC CCGTGGACTC GTTTGGAAAG CTCTCTCAGA TCATTCACAG TCCCAGCTTC  601 CACGAGAGAT GA Oreochromis niloticus (tilapia) FGF19 gene coding sequence (1-208)(SEQ ID NO: 324) (Ensembl accession no. ENSONIT00000022816, which is hereby incorporated by reference in its entirety)   55                                                            ATGCTG   61 CTGCTCCTCA TCGTATCCAT TGTCAATATG CTTTTTGGTG TTGGAATGGT TTGCATGCCC  121 CTGTCAGACA ACGGGCCCCA CATCGCCCAC GGCTGGGCCC AGGTGGTCCG GCTCAGGCAC  181 CTTTACGCCA CCAGACCGGG AATGCACCTG CTGATCAGTG AGGGTGGACA GATCCGTGGT  241 TCTGCCGTCC AGACTCTGCA CAGCCTAATG GAGATTCGTC CAGTCGGTCC AGGCCGTGTT  301 GTCATCAGAG GGGTAGCAAC CGCAAGGTTT CTCTGCATAG AAGACGACGG CACACTGTAC  361 TCATCGCACG CCTACAGCAG AGAGGACTGC ATCTTCAGAG AGCAGATCTT GCCAGATGGG  421 TACAACATCT ACATCTCTGA CAGACATGGA GTCCTGCTCA GTCTGGGAAA CCACCGGCAA  481 AGACTGCAGG GCTTAGACCG AGGAGATCCA GCCCTGGCCC AGTTCCTCCC CAGGATCAGC  541 ACTCTGAATC AAATCCCTTC CCCTGGGGCA AACATCGGTG ACCACATGAA AGTAGCAAAA  601 ACAGAAGAAC CTGTGGACAC AATAGATTCA TTTGGAAAGT TCTCTCAGAT CATTGACAGT  607 CCCAGCTTCC ATAAGAGATG A Meleagris gallopavo (turkey) FGF19 gene coding sequence (1-216, excluding 1-70)(SEQ ID NO: 325) (Ensembl accession no. ENSMGAT00000011114, which is hereby incorporated by reference in its entirety)    1 GTAGGCAATC AATCACCACA GAGCATCCTT GAAATAACTG CTGTTGATGT CGGGATCGTC   61 GCTATCAAGG GCTTGTTCTC TGGCAGATAC CTGGCCATGA ACAAAAGGGG CAGGCTTTAT  121 GCATCACTCA GCTATTCCAT TGAGGACTGT TCCTTTGAAG AGGAGATTCG TCCAGATGGC  181 TATAACGTGT ATAAATCAAA GAAATACGGA ATATCAGTGT CTTTGAGCAG TGCCAAACAA  241 AGACAACAAT TCAAAGGAAA AGATTTTCTC CCACTGTCTC ACTTCTTACC CATGATCAAC  301 ACTGTGCCAG TGGAGGTGAC AGACTTTGGT GAATACGGTG ATTACAGCCA GGCTTTTGAG  361 CCAGAGGTCT ACTCATCGCC TCTCGAAACG GACAGCATGG ATCCCTTTGG GATCACTTCC  421 AAACTGTCTC CAGTGAAGAG CCCCAGCTTT CAGAAA Papio anubis (olive baboon) FGF19 gene coding sequence (1-216)(SEQ ID NO: 326) (GenBank accession no. XM_003909422, which is hereby incorporated by reference in its entirety)  758                                         ATG AGGAGCGGGT GTGTGGTGGT  781 CCACGCCTGG ATCCTGGCCA GCCTCTGGCT GGCCGTGGCC GGGCGTCCCC TCGCCTTCTC  841 GGACGCGGGG CCCCACGTGC ACTACGGCTG GGGCGACCCC ATCCGCCTGC GGCACCTGTA  901 CACCTCCGGC CCCCACGGGC TCTCCAGCTG CTTCCTGCGC ATCCGCACCG ACGGCGTCGT  961 GGACTGCGCG CGGGGCCAAA GCGCGCACAG TTTGCTGGAG ATCAAGGCAG TAGCTCTGCG 1021 GACCGTGGCC ATCAAGGGCG TGCACAGCGT GCGGTACCTC TGCATGGGCG CCGACGGCAA 1081 GATGCAGGGG CTGCTTCAGT ACTCAGAGGA AGACTGTGCT TTCGAGGAGG AGATCCGCCC 1141 TGATGGCTAC AATGTATACC GATCCCAGAA GCACCGCCTC CCGGTCTCCC TGAGCAGTGC 1201 CAAACAGCGG CAGCTGTACA AGAACAGAGG CTTTCTTCCG CTGTCTCATT TCCTGCCCAT 1261 GCTGCCCATG GCCCCAGAGG AGCCTGAGGA CCTCAGGGGC CCCTTGGAAT CTGACATGTT 1321 CTCTTCGCCC CTGGAGACTG ACAGCATGGA CCCATTTGGG CTTGTCACCG GACTGGAGGC 1381 GGTGAGGAGT CCCAGCTTTG AGAAATAA Saimiri boliviensis boliviensis (Bolivian squirrel monkey) FGF19 gene coding sequence (1-216)(SEQ ID NO: 327) (GenBank accession no. XM_003941165, which is hereby incorporated by reference in its entirety)  231                                                        ATGCGGAGCG  241 GGTGTGTGGT GGTCCACGCC TGGATCCTGG CTGGCCTCTG GCTGGCTGTG GTCGGGCGCC  301 CCCTCGCCTT CTCCGATGCG GGGCCGCATG TGCATTACGG CTGGGGCGAC CCCATTCGCC  361 TGCGGCACCT GTACACCTCC AGCCCCCACG GCCTCTCCAG CTGCTTCCTG CGCATCCGCA  421 GCGACGGCGT CGTGGACTGC GCGCGGGGCC AGAGCGCGCA CAGTTTGCTG GAGATCAAGG  481 CAGTCGCTCT AAGGACCGTG GCCATCAAGG GCGTGCACAG CTCGCGGTAC CTCTGCATGG  541 GCGCCGACGG CAGGCTGCAG GGGCTGTTCC AGTACTCGGA GGAAGACTGT GCTTTCGAGG  601 AGGAGATCCG CCCCGACGGC TACAATGTGT ACCTATCCGA GAAGCACCGC CTCCCGGTCT  661 CCCTGAGCAG CGCCAAACAG CGGCAGCTGT ACAAGAAACG AGGCTTTCTT CCGCTGTCCC  721 ATTTCCTGCC CATGCTGCCC AGAGCCCCAG AGGAGCCTGA TGACCTCAGG GGCCACTTGG  781 AATCTGACGT GTTCTCTTCA CCCCTGGAGA CTGATAGCAT GGACCCATTT GGGCTTGTCA  841 CGGGACTGGA GGCGGTGAAC AGTCCCAGCT TTGAGAAGTA A Pteropus alecto (black flying fox) FGF19 gene coding sequence (1-216) (SEQ ID NO: 328) (generated using SMS Reverse Translate tool on the ExPASy Bioinformatics Resource website (www.expasy.org)    1 ATGCGCAGCC CGTGCGCGGT GGCGCGCGCG CTGGTGCTGG CGGGCCTGTG GCTGGCGAGC   61 GCGGCGGGCC CGCTGGCGCT GAGCGATGCG GGCCCGCATG TGCATTATGG CTGGGGCGAA  121 GCGATTCGCC TGCGCCATCT GTATACCGCG GGCCCGCATG GCCCGAGCAG CTGCTTTCTG  181 CGCATTCGCG CGGATGGCGC GGTGGATTGC GCGCGCGGCC AGAGCGCGCA TAGCCTGGTG  241 GAAATTCGCG CGGTGGCGCT GCGCAACGTG GCGATTAAAG GCGTGCATAG CGTGCGCTAT  301 CTGTGCATGG GCGCGGATGG CCGCATGCTG GGCCTGCTGC AGTATAGCGC GGATGATTGC  361 GCGTTTGAAG AAGAAATTCG CCCGGATGGC TATAACGTGT ATCATAGCAA AAAACATCAT  421 CTGCCGGTGA GCCTGAGCAG CGCGAAACAG CGCCAGCTGT ATAAAGATCG CGGCTTTCTG  481 CCGCTGAGCC ATTTTCTGCC GATGCTGCCG CGCAGCCCGA CCGAACCGGA AAACTTTGAA  541 GATCATCTGG AAGCGGATAC CTTTAGCAGC CCGCTGGAAA CCGATGATAT GGATCCGTTT  601 GGCATTGCGA GCAAACTGGG CCTGGAAGAA AGCCCGAGCT TTCAGAAA Myotis davidii (David's myotis) FGF19 gene coding sequence (1-245)(SEQ ID NO: 329) (generated using SMS Reverse Translate tool on the ExPASy Bioinformatics Resource website (www.expasy.org))    1 ATGAGCGGCC AGAACAGCGG CCGCCATGGC AGCCGCCCGG GCCTGGATGA AGAACCGGAA   61 CCGGGCCCGC TGGAACTGCG CGCGCTGGGC AGCACCCGCG CGGATCCGCA GCTGTGCGAT  121 TTTCTGGAAA ACCATTTTCT GGGCTATACC TGCCTGGAAC TGGATATTTG CCTGGCGACC  181 TATCTGGGCG TGAGCCATTG GGGCGAAAGC ATTCGCCTGC GCCATCTGTA TACCAGCGGC  241 CCGCATGGCC CGAGCAGCTG CTTTCTGCGC ATTCGCGTGG ATGGCGCGGT GGATTGCGCG  301 CGCGGCCAGA GCGCGCATAG CCTGGTGGAA ATTCGCGCGG TGGCGCTGCG CAAAGTGGCG  361 ATTAAAGGCG TGCATAGCGC GCTGTATCTG TGCATGGAAG GCGATGGCCG CATGCGCGGC  421 CTGCCGCAGT TTAGCCCGGA AGATTGCGCG TTTGAAGAAG AAATTCGCCC GGATGGCTAT  481 AACGTGTATC GCAGCCAGAA ACATCAGCTG CCGGTGAGCC TGAGCAGCGC GCGCCAGCGC  541 CAGCTGTTTA AAGCGCGCGG CTTTCTGCCG CTGAGCCATT TTCTGCCGAT GCTGCCGAGC  601 AGCCCGGCGG AACCGGTGCA TCGCGAACGC CCGCTGGAAC CGGATGCGTT TAGCAGCCCG  661 CTGGAAACCG ATAGCATGGA TCCGTTTGGC ATTGCGAACA ACCTGCGCCT GGTGAAAAGC  721 CCGAGCTTTC AGAAA Tupaia chinensis (Chinese tree shrew) FGF19 gene coding sequence (1-257,excluding 13-19)(SEQ ID NO: 330) (generated using SMS Reverse Translate tool on the ExPASy Bioinformatics Resource website (www.expasy.org))    1 ATGCGCCGCA CCTGGAGCGG CTTTGCGGTG GCGACC---- ---------- ----CGCGCG   61 GGCAGCCCGC TGGCGCTGGC GGATGCGGGC CCGCATGTGA ACTATGGCTG GGATGAAAGC  121 ATTCGCCTGC GCCATCTGTA TACCGCGAGC CTGCATGGCA GCACCAGCTG CTTTCTGCGC  181 ATTCGCGATG ATGGCAGCGT GGGCTGCGCG CGCGGCCAGA GCATGCATAG CCTGCTGGAA  241 ATTAAAGCGG TGGCGCTGCA GACCGTGGCG ATTAAAGGCG TGTATAGCGT GCGCTATCTG  301 TGCATGGATA CCGATGGCCG CATGCAGGGC CTGCCGCAGT ATAGCGAAGA AGATTGCACC  361 TTTGAAGAAG AAATTCGCAG CGATGGCCAT AACGTGTATC GCAGCAAAAA ACATGGCCTG  421 CCGGTGAGCC TGAGCAGCGC GAAACAGCGC CAGCTGTATA AAGGCCGCGG CTTTCTGAGC  481 CTGAGCCATT TTCTGCTGAT GATGCCGAAA ACCAGCGCGG GCCCGGGCAA CCCGCGCGAT  541 CAGCGCAACC CGCGCGATCA GCGCGATCCG AACACCTTTA GCCTGCCGCT GGAAACCGAT  601 AGCATGGATC CGTTTGGCAT GACCACCCGC CATGGCCTGC TGCTGGATAG CTGCTGCGCG  661 AGCCTGGTGC TGCTGAACAT TAGCACCGAT GGCGAATTTA GCCCGTATGG CAACATTCTG  721 CGCCCGAGCT TTCGCTTTAA ACTGTTTAAA ATGAAAAAAG TGACCAAC Heterocephalus glaber (naked mole-rat) FGF19 gene coding sequence (1-209)(SEQ ID NO: 331) (generated using SMS Reverse Translate tool onthe ExPASy Bioinformatics Resource website (www.expasy.org))    1 ATGCGCTTTA GCAAAAGCAC CTGCGGCTTT TTTAACCATC AGCGCCTGCA GGCGCTGTGG   61 CTGAGCCTGA GCAGCGTGAA ATGGGTGCTG GATGCGGCGG TGGAAGGCCG CCCGATTCGC  121 CTGCGCCATC TGTATGCGGC GGGCCCGTAT GGCCGCAGCC GCTGCTTTCT GCGCATTCAT  181 ACCGATGGCG CGGTGGATTG CGTGGAAGAA CAGAGCGAAC ATTGCCTGCT GGAAATTCGC  241 GCGGTGGCGC TGGAAACCGT GGCGATTAAA GATATTAACA GCGTGCGCTA TCTGTGCATG  301 GGCCCGGATG GCCGCATGCA GGGCCTGCCG TGGTATAGCG AAGAAGATTG CGCGTTTAAA  361 GAAGAAATTA GCTATCCGGG CTATAGCGTG TATCGCAGCC AGAAACATCA TCTGCCGATT  421 GTGCTGAGCA GCGTGAAACA GCGCCAGCAG TATCAGAGCA AAGGCGTGGT GCCGCTGAGC  481 TATTTTCTGC CGATGCTGCC GAAAGCGAGC GTGGAACCGG GCGATGAAGA AGAAAGCGCG  541 TTTAGCCTGC CGCTGAAAAC CGATAGCATG GATCCGTTTG GCATGGCGAG CGAAATTGGC  601 CTGGCGAAAA GCCCGAGCTT TCAGAAA 

In one embodiment of the present invention, the chimeric protein mayinclude one or more substitutions for or additions of amino acids fromanother FGF. In one embodiment, the C-terminal portion from FGF19includes a modification that includes a substitution for or addition ofamino acid residues from an FGF21 (including a human FGF21 and orthologsof human FGF21). In one embodiment the FGF21 is a human FGF21 proteinhaving an amino acid sequence of SEQ ID NO: 332 (GenBank Accession No.NP_061986, which is hereby incorporated by reference in its entirety) ora portion thereof, as follows:

1 MDSDETGFEH SGLWVSVLAG LLLGACQAHP IPDSSPLLQF GGQVRQRYLY TDDAQQTEAH 61LEIREDGTVG GAADQSPESL LQLKALKPGV IQILGVKTSR FLCQRPDGAL YGSLHFDPEA 121CSFRELLLED GYNVYQSEAH GLPLHLPGNK SPHRDPAPRG PARFLPLPGL PPALPEPPGI 181LAPQPPDVGS SDPLSMVGPS QGRSPSYASExemplary substitutions and additions of such residues are shown in FIG.13.

In one embodiment, the C-terminal portion from FGF19 comprises amodification that includes a substitution of amino acid residues from anFGF21. In one embodiment, the modification comprises a substitution foror addition of amino acid residues 168 to 209 of SEQ ID NO: 332 (FGF21).In one embodiment, the modification is a substitution of amino acidresidues from SEQ ID NO: 332 (FGF21) for corresponding amino acidresidues of SEQ ID NO: 233. The corresponding residues of FGFs may beidentified by sequence analysis and/or structural analysis. See FIGS. 2,11, and 13. In one embodiment, the modification includes a substitutionof a contiguous stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42 amino acid residues168 to 209 of SEQ ID NO: 332 (FGF21) for the corresponding contiguousstretch of amino acid residues of SEQ ID NO: 233. In one embodiment,amino acid residues 169 to 173, 169 to 196, or 169 to 203 of SEQ ID NO:233 are substituted with the corresponding amino acid residues selectedfrom the sequence comprising amino acid residues 168 to 209 of SEQ IDNO: 332 (FGF21).

In one embodiment, the modification includes a substitution of one ormore individual amino acid residues from residues 168 to 209 of SEQ IDNO: 332 (FGF21) for the corresponding amino acid residues of SEQ ID NO:233. In one embodiment, the C-terminal portion includes substitutions ofone or more of amino acid residues 169, 170, 171, 172, 174, 175, 183,184, 185, 186, 187, 188, 189, 190, 192, 193, 194, 195, 197, 200, 201,202, 206, 207, 208, 209, 214, 215, or 216 of SEQ ID NO: 1 for thecorresponding amino acid residues of SEQ ID NO: 332 (FGF21).

In one embodiment of the present invention, the C-terminal portion fromFGF19 includes a modification that includes a deletion of amino acidresidues that are absent in the corresponding C-terminal portion fromFGF21. As shown in FIG. 13, FGF19 residues that are absent in thecorresponding C-terminal portion of FGF21 may be identified by sequenceanalysis and/or structural analysis. In one embodiment, the modificationcomprises a deletion of amino acid residues selected from residues 204to 216, 197 to 216, 174 to 216, or 169 to 216 of SEQ ID NO: 233. In oneembodiment, the modification comprises a deletion corresponding to aminoacid residue 204 of SEQ ID NO: 233. In one embodiment, the modificationincludes a deletion of amino acid residues 178, 179, 180, 181, and/or182 of SEQ ID NO: 233 individually or in combination.

Chimeric proteins according to the present invention may be isolatedproteins or polypeptides. The isolated chimeric proteins of the presentinvention may be prepared for use in the above described methods of thepresent invention using standard methods of synthesis known in the art,including solid phase peptide synthesis (Fmoc or Boc strategies) orsolution phase peptide synthesis. Alternatively, peptides of the presentinvention may be prepared using recombinant expression systems.

Chimeric proteins according to the present invention may be isolatedproteins or polypeptides. The isolated chimeric proteins of the presentinvention may be prepared for use in the above described methods of thepresent invention using standard methods of synthesis known in the art,including solid phase peptide synthesis (Fmoc or Boc strategies) orsolution phase peptide synthesis. Alternatively, peptides of the presentinvention may be prepared using recombinant expression systems.

In one embodiment, the chimeric protein of the present inventionincludes the amino acid sequence of SEQ ID NO: 333, SEQ ID NO: 334, SEQID NO: 335, or SEQ ID NO: 336, as shown in Table 9.

TABLE 9  Description of Chimeric Protein SequenceAmino acid sequence of a SEQ ID NO: 333 FGF1/FGF19 chimera composedMAEGEITTFT ALTEKFNLPP GNYKKPKLLY of residues M1 to L150 of humanCSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ FGF1 harboringLSAESVGEVY IKSTETGQYL AMDTDGLLYG K127D/K128Q/K133V tripleSQTPNEECLF LERLEENHYN TYISKKHAEK mutation (bold) and residuesNWFVGLDQNG SCVRGPRTHY GQKAILFLPL L169 to K216 of human FGF19LPMVPEEPED LRGHLESDMF SSPLETDSMD (bold) PFGLVTGLEA VRSPSFEKAmino acid sequence of a SEQ ID NO: 334 FGF1/FGF19 chimera composed                          KPKLLY of residues K25 to L150 of humanCSNGGHFLRI LPDGTVDGTR DRSDQHIQLQ FGF1 harboringLSAESVGEVY IKSTETGQYL AMDTDGLLYG K127D/K128Q/K133V tripleSQTPNEECLF LERLEENHYN TYISKKHAEK mutation (bold) and residuesNWFVGLDQNG SCVRGPRTHY GQKAILFLPL L169 to K216 of human FGF19LPMVPEEPED LRGHLESDMF SSPLETDSMD (bold) PFGLVTGLEA VRSPSFEKAmino acid sequence of a SEQ ID NO: 335 FGF2/FGF19 chimera composedMAAGSITTLP ALPEDGGSGA FPPGHFKDPK of residues M1 to M151 of humanRLYCKNGGFF LRIHPDGRVD GVREKSDPHI FGF2 harboringKLQLQAEERG VVSIKGVCAN RYLAMKEDGR K128D/R129Q/K134V tripleLLASKCVTDE CFFFERLESN NYNTYRSRKY mutation (bold) and residuesTSWYVALDQT GQYVLGSKTG PGQKAILFLP L169 to K216 of human FGF19MLPMVPEEPE DLRGHLESDM FSSPLETDSM (bold) DPFGLVTGLE AVRSPSFEKAmino acid sequence of a SEQ ID NO: 336 FGF2/FGF19 chimera composed                          HFKDPK of residues H25 to M151 ofRLYCKNGGFF LRIHPDGRVD GVREKSDPHI human FGF2 harboringKLQLQAEERG VVSIKGVCAN RYLAMKEDGR K128D/R129Q/K134V tripleLLASKCVTDE CFFFERLESN NYNTYRSRKY mutation (bold) and residuesTSWYVALDQT GQYVLGSKTG PGQKAILFLP L169 to K216 of human FGF19MLPMVPEEPE DLRGHLESDM FSSPLETDSM (bold) DPFGLVTGLE AVRSPSFEK

Chimeric proteins according to the present invention may be isolatedproteins or polypeptides. The isolated chimeric proteins of the presentinvention may be prepared for use in accordance with the presentinvention using standard methods of synthesis known in the art,including solid phase peptide synthesis (Fmoc or Boc strategies) orsolution phase peptide synthesis. Alternatively, peptides of the presentinvention may be prepared using recombinant expression systems.

Accordingly, another aspect of the present invention relates to anisolated nucleic acid molecule encoding a chimeric protein according tothe present invention. In one embodiment, the nucleic acid moleculecomprises the nucleotide sequence of SEQ ID NO: 337, SEQ ID NO: 338, SEQID NO: 339, or SEQ ID NO: 340, as shown in Table 10.

TABLE 10  Description of Chimeric Protein SequenceNucleotide sequence of a SEQ ID NO: 337 FGF1/FGF19 chimera composedATGGCTGAAG GGGAAATCAC CACCTTCACA of residues M1 to L150 of humanGCCCTGACCG AGAAGTTTAA TCTGCCTCCA FGF1 harboringGGGAATTACA AGAAGCCCAA ACTCCTCTAC K127D/K128Q/K133V tripleTGTAGCAACG GGGGCCACTT CCTGAGGATC mutation (bold) and residuesCTTCCGGATG GCACAGTGGA TGGGACAAGG L169 to K216 of human FGF19GACAGGAGCG ACCAGCACAT TCAGCTGCAG (bold) CTCAGTGCGG AAAGCGTGGG GGAGGTGTATATAAAGAGTA CCGAGACTGG CCAGTACTTG GCCATGGACA CCGACGGGCT TTTATACGGCTCACAGACAC CAAATGAGGA ATGTTTGTTC CTGGAAAGGC TGGAGGAGAA CCATTACAACACCTATATAT CCAAGAAGCA TGCAGAGAAG AATTGGTTTG TTGGCCTCGA TCAGAATGGGAGCTGCGTTC GCGGTCCTCG GACTCACTAT GGCCAGAAAG CAATCTTGTT TCTCCCCCTGCTGCCCATGG TCCCAGAGGA GCCTGAGGAC CTCAGGGGCC ACTTGGAATC TGACATGTTCTCTTCGCCCC TGGAGACCGA CAGCATGGAC CCATTTGGGC TTGTCACCGG ACTGGAGGCCGTGAGGAGTC CCAGCTTTGA GAAG Nucleotide sequence of a SEQ ID NO: 338FGF1/FGF19 chimera composed              AAGCCCAA ACTCCTCTACof residues K25 to L150 of human TGTAGCAACG GGGGCCACTT CCTGAGGATCFGF1 harboring CTTCCGGATG GCACAGTGGA TGGGACAAGG K127D/K128Q/K133V tripleGACAGGAGCG ACCAGCACAT TCAGCTGCAG mutation (bold) and residuesCTCAGTGCGG AAAGCGTGGG GGAGGTGTAT L169 to K216 of human FGF19ATAAAGAGTA CCGAGACTGG CCAGTACTTG (bold) GCCATGGACA CCGACGGGCT TTTATACGGCTCACAGACAC CAAATGAGGA ATGTTTGTTC CTGGAAAGGC TGGAGGAGAA CCATTACAACACCTATATAT CCAAGAAGCA TGCAGAGAAG AATTGGTTTG TTGGCCTCGA TCAGAATGGGAGCTGCGTTC GCGGTCCTCG GACTCACTAT GGCCAGAAAG CAATCTTGTT TCTCCCCCTGCTGCCCATGG TCCCAGAGGA GCCTGAGGAC CTCAGGGGCC ACTTGGAATC TGACATGTTCTCTTCGCCCC TGGAGACCGA CAGCATGGAC CCATTTGGGC TTGTCACCGG ACTGGAGGCCGTGAGGAGTC CCAGCTTTGA GAAG Nucleotide sequence of a SEQ ID NO: 339FGF2/FGF19 chimera composed                   ATG GCAGCCGGGAof residues M1 to M151 of human GCATCACCAC GCTGCCCGCC TTGCCCGAGGFGF2 harboring ATGGCGGCAG CGGCGCCTTC CCGCCCGGCC K128D/R129Q/K134V tripleACTTCAAGGA CCCCAAGCGG CTGTACTGCA mutation (bold) and residuesAAAACGGGGG CTTCTTCCTG CGCATCCACC L169 to K216 of human FGF19CCGACGGCCG AGTTGACGGG GTCCGGGAGA (bold) AGAGCGACCC TCACATCAAG CTACAACTTCAAGCAGAAGA GAGAGGAGTT GTGTCTATCA AAGGAGTGTG TGCTAACCGT TACCTGGCTATGAAGGAAGA TGGAAGATTA CTGGCTTCTA AATGTGTTAC GGATGAGTGT TTCTTTTTTGAACGATTGGA ATCTAATAAC TACAATACTT ACCGGTCAAG GAAATACACC AGTTGGTATGTGGCACTGGA TCAGACTGGG CAGTATGTTC TTGGATCCAA AACAGGACCT GGGCAGAAAGCTATACTTTT TCTTCCAATG CTGCCCATGG TCCCAGAGGA GCCTGAGGAC CTCAGGGGCCACTTGGAATC TGACATGTTC TCTTCGCCCC TGGAGACCGA CAGCATGGAC CCATTTGGGCTTGTCACCGG ACTGGAGGCC GTGAGGAGTC CCAGCTTTGA GAAGNucleotide sequence of a SEQ ID NO: 340 FGF2/FGF19 chimera composed                               C of residues H25 to M151 ofACTTCAAGGA CCCCAAGCGG CTGTACTGCA human FGF2 harboringAAAACGGGGG CTTCTTCCTG CGCATCCACC K128D/R129Q/K134V tripleCCGACGGCCG AGTTGACGGG GTCCGGGAGA mutation (bold) and residuesAGAGCGACCC TCACATCAAG CTACAACTTC L169 to K216 of human FGF19AAGCAGAAGA GAGAGGAGTT GTGTCTATCA (bold) AAGGAGTGTG TGCTAACCGT TACCTGGCTATGAAGGAAGA TGGAAGATTA CTGGCTTCTA AATGTGTTAC GGATGAGTGT TTCTTTTTTGAACGATTGGA ATCTAATAAC TACAATACTT ACCGGTCAAG GAAATACACC AGTTGGTATGTGGCACTGGA TCAGACTGGG CAGTATGTTC TTGGATCCAA AACAGGACCT GGGCAGAAAGCTATACTTTT TCTTCCAATG CTGCCCATGG TCCCAGAGGA GCCTGAGGAC CTCAGGGGCCACTTGGAATC TGACATGTTC TCTTCGCCCC TGGAGACCGA CAGCATGGAC CCATTTGGGCTTGTCACCGG ACTGGAGGCC GTGAGGAGTC CCAGCTTTGA GAAG

Another aspect of the present invention relates to a nucleic acidconstruct comprising a nucleic acid molecule encoding a chimeric proteinaccording to the present invention, a 5′ DNA promoter sequence, and a 3′terminator sequence. The nucleic acid molecule, the promoter, and theterminator are operatively coupled to permit transcription of thenucleic acid molecule.

Also encompassed are vectors or expression vectors comprising suchnucleic acid molecules and host cells comprising such nucleic acidmolecules. Nucleic acid molecules according to the present invention canbe expressed in a host cell, and the encoded polynucleotides isolated,according to techniques that are known in the art.

Generally, the use of recombinant expression systems involves insertingthe nucleic acid molecule encoding the amino acid sequence of thedesired peptide into an expression system to which the molecule isheterologous (i.e., not normally present). One or more desired nucleicacid molecules encoding a peptide of the invention may be inserted intothe vector. When multiple nucleic acid molecules are inserted, themultiple nucleic acid molecules may encode the same or differentpeptides. The heterologous nucleic acid molecule is inserted into theexpression system or vector in proper sense (5′→3′) orientation relativeto the promoter and any other 5′ regulatory molecules, and correctreading frame.

The preparation of the nucleic acid constructs can be carried out usingstandard cloning procedures well known in the art as described by JosephSambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL (Cold SpringsHarbor 1989). U.S. Pat. No. 4,237,224 to Cohen and Boyer, which ishereby incorporated by reference in its entirety, describes theproduction of expression systems in the form of recombinant plasmidsusing restriction enzyme cleavage and ligation with DNA ligase. Theserecombinant plasmids are then introduced by means of transformation andreplicated in a suitable host cell.

A variety of genetic signals and processing events that control manylevels of gene expression (e.g., DNA transcription and messenger RNA(“mRNA”) translation) can be incorporated into the nucleic acidconstruct to maximize protein production. For the purposes of expressinga cloned nucleic acid sequence encoding a desired protein, it isadvantageous to use strong promoters to obtain a high level oftranscription. Depending upon the host system utilized, any one of anumber of suitable promoters may be used. For instance, when cloning inE. coli, its bacteriophages, or plasmids, promoters such as the T7 phagepromoter, lac promoter, trp promoter, recA promoter, ribosomal RNApromoter, the P_(R) and P_(L) promoters of coliphage lambda and others,including but not limited, to lacUV5, ompF, bla, lpp, and the like, maybe used to direct high levels of transcription of adjacent DNA segments.Additionally, a hybrid trp-lacUV5 (tac) promoter or other E. colipromoters produced by recombinant DNA or other synthetic DNA techniquesmay be used to provide for transcription of the inserted gene. Commonpromoters suitable for directing expression in mammalian cells include,without limitation, SV40, MMTV, metallothionein-1, adenovirus Ela, CMV,immediate early, immunoglobulin heavy chain promoter and enhancer, andRSV-LTR.

There are other specific initiation signals required for efficient genetranscription and translation in prokaryotic cells that can be includedin the nucleic acid construct to maximize protein production. Dependingon the vector system and host utilized, any number of suitabletranscription and/or translation elements, including constitutive,inducible, and repressible promoters, as well as minimal 5′ promoterelements, enhancers or leader sequences may be used. For a review onmaximizing gene expression see Roberts and Lauer, “Maximizing GeneExpression On a Plasmid Using Recombination In Vitro,” Methods inEnzymology 68:473-82 (1979), which is hereby incorporated by referencein its entirety.

A nucleic acid molecule encoding an isolated protein of the presentinvention, a promoter molecule of choice, including, without limitation,enhancers, and leader sequences; a suitable 3′ regulatory region toallow transcription in the host, and any additional desired components,such as reporter or marker genes, are cloned into the vector of choiceusing standard cloning procedures in the art, such as described inJoseph Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL (ColdSprings Harbor 1989); Frederick M. Ausubel, SHORT PROTOCOLS IN MOLECULARBIOLOGY (Wiley 1999); and U.S. Pat. No. 4,237,224 to Cohen and Boyer,which are hereby incorporated by reference in their entirety.

Once the nucleic acid molecule encoding the protein has been cloned intoan expression vector, it is ready to be incorporated into a host.Recombinant molecules can be introduced into cells, without limitation,via transfection (if the host is a eukaryote), transduction,conjugation, mobilization, or electroporation, lipofection, protoplastfusion, mobilization, or particle bombardment, using standard cloningprocedures known in the art, as described by JOSEPH SAMBROOK et al.,MOLECULAR CLONING: A LABORATORY MANUAL (Cold Springs Harbor 1989), whichis hereby incorporated by reference in its entirety.

A variety of suitable host-vector systems may be utilized to express therecombinant protein or polypeptide. Primarily, the vector system must becompatible with the host used. Host-vector systems include, withoutlimitation, the following: bacteria transformed with bacteriophage DNA,plasmid DNA, or cosmid DNA; microorganisms such as yeast containingyeast vectors; mammalian cell systems infected with virus (e.g.,vaccinia virus, adenovirus, etc.); insect cell systems infected withvirus (e.g., baculovirus); and plant cells infected by bacteria.

Purified proteins may be obtained by several methods readily known inthe art, including ion exchange chromatography, hydrophobic interactionchromatography, affinity chromatography, gel filtration, and reversephase chromatography. The protein is preferably produced in purifiedform (preferably at least about 80% or 85% pure, more preferably atleast about 90% or 95% pure) by conventional techniques. Depending onwhether the recombinant host cell is made to secrete the protein intogrowth medium (see U.S. Pat. No. 6,596,509 to Bauer et al., which ishereby incorporated by reference in its entirety), the protein can beisolated and purified by centrifugation (to separate cellular componentsfrom supernatant containing the secreted protein) followed by sequentialammonium sulfate precipitation of the supernatant. The fractioncontaining the protein is subjected to gel filtration in anappropriately sized dextran or polyacrylamide column to separate theprotein of interest from other proteins. If necessary, the proteinfraction may be further purified by HPLC.

Another aspect of the present invention relates to a pharmaceuticalcomposition that includes a chimeric protein according to the presentinvention and a pharmaceutically acceptable carrier.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers which are nontoxic to the cell or mammalbeing exposed thereto at the dosages and concentrations employed. Oftenthe physiologically acceptable carrier is an aqueous pH bufferedsolution. Examples of physiologically acceptable carriers includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

The term “pharmaceutically acceptable” means it is, within the scope ofsound medical judgment, suitable for use in contact with the cells ofhumans and lower animals without undue toxicity, irritation, allergicresponse, and the like, and is commensurate with a reasonablebenefit/risk ratio.

In one embodiment, the pharmaceutical composition includes anorganotropic targeting agent. In one embodiment, the targeting agent iscovalently linked to the chimeric protein via a linker that is cleavedunder physiological conditions.

Chimeric and/or modified proteins according to the present invention mayalso be modified using one or more additional or alternative strategiesfor prolonging the in vivo half-life of the protein. One such strategyinvolves the generation of D-peptide chimeric proteins, which consist ofunnatural amino acids that are not cleaved by endogenous proteases.Alternatively, the chimeric and/or modified proteins may be fused to aprotein partner that confers a longer half-life to the protein upon invivo administration. Suitable fusion partners include, withoutlimitation, immunoglobulins (e.g., the Fc portion of an IgG), humanserum albumin (HAS) (linked directly or by addition of the albuminbinding domain of streptococcal protein G), fetuin, or a fragment of anyof these. The chimeric and/or modified proteins may also be fused to amacromolecule other than protein that confers a longer half-life to theprotein upon in vivo administration. Suitable macromolecules include,without limitation, polyethylene glycols (PEGs). Methods of conjugatingproteins or peptides to polymers to enhance stability for therapeuticadministration are described in U.S. Pat. No. 5,681,811 to Ekwuribe,which is hereby incorporated by reference in its entirety. Nucleic acidconjugates are described in U.S. Pat. No. 6,528,631 to Cook et al., U.S.Pat. No. 6,335,434 to Guzaev et al., U.S. Pat. No. 6,235,886 toManoharan et al., U.S. Pat. No. 6,153,737 to Manoharan et al., U.S. Pat.No. 5,214,136 to Lin et al., or U.S. Pat. No. 5,138,045 to Cook et al.,which are hereby incorporated by reference in their entirety.

The pharmaceutical composition according to the present invention can beformulated for administration orally, parenterally, subcutaneously,intravenously, intramuscularly, intraperitoneally, by intranasalinstillation, by implantation, by intracavitary or intravesicalinstillation, intraocularly, intraarterially, intralesionally,transdermally, or by application to mucous membranes. The formulationsmay conveniently be presented in unit dosage form and may be prepared byany of the methods well known in the art of pharmacy.

Another aspect of the present invention relates to a method for treatinga subject suffering from a disorder. This method involves selecting asubject suffering from the disorder and administering the pharmaceuticalcomposition according to the present invention to the selected subjectunder conditions effective to treat the disorder. In one embodiment thedisorder is diabetes, obesity, or metabolic syndrome.

Accordingly, another aspect of the present invention relates to a methodfor treating a subject suffering from a disorder. This method involvesselecting a subject suffering from the disorder. The method alsoinvolves providing a chimeric FGF protein, where the chimeric FGFprotein includes an N-terminus coupled to a C-terminus. The N-terminusincludes a portion of a paracrine FGF and the C-terminus includes aC-terminal portion of FGF19. The portion of the paracrine FGF ismodified to decrease binding affinity for heparin and/or heparan sulfatecompared to the portion without the modification. This method alsoinvolves administering a therapeutically effective amount of thechimeric FGF protein to the selected subject under conditions effectiveto treat the disorder.

The portion of the paracrine FGF may also be modified to alterreceptor-binding specificity and/or receptor-binding affinity comparedto the portion without the modification. Suitable chimeric proteins foruse in accordance with this aspect of the present invention aredescribed above and throughout the present application.

In one embodiment, the selected subject is a mammal. In one embodiment,the selected subject is a human. In another embodiment, the selectedsubject is a rodent.

In one embodiment, the selected subject is in need of increasedFGF19-βKlotho-FGF receptor (“FGFR”) complex formation.

In one embodiment, the disorder is a selected from diabetes, obesity,and metabolic syndrome. As used herein, diabetes includes type Idiabetes, type II diabetes, gestational diabetes, and drug-induceddiabetes. In yet another embodiment, the subject has obesity. In yetanother embodiment, the subject has metabolic syndrome.

The chimeric protein of the present invention or pharmaceuticalcomposition thereof can be used to treat a number of conditions. In oneembodiment, the condition is one which the therapeutic outcome includesa decrease in blood glucose, a decrease in blood fructosamine, anincrease in energy expenditure, an increase in fat utilization, adecrease in body weight, a decrease in body fat, a decrease intriglycerides, a decrease in free fatty acids, an increase in fatexcretion, an improvement, or even a preservation, of pancreatic 3-cellfunction and mass, a decrease in total blood cholesterol, a decrease inblood low-density lipoprotein cholesterol, an increase in bloodhigh-density lipoprotein cholesterol, an increase in blood adiponectin,an increase in insulin sensitivity, an increase in leptin sensitivity, adecrease in blood insulin, a decrease in blood leptin, a decrease inblood glucagon, an increase in glucose uptake by adipocytes, a decreasein fat accumulation in hepatocytes, and/or an increase in fat oxidationin hepatocytes. Each of these parameters can be measured by standardmethods, for example, by measuring oxygen consumption to determinemetabolic rate, using scales to determine weight, and measuring leanbody mass composition or mass to determine fat. Moreover, the presenceand amount of triglycerides, free fatty acids, glucose and leptin can bedetermined by standard methods (e.g., blood test).

Additional conditions that are treatable in accordance with the presentinvention include one or more of type 1 diabetes, type 2 diabetes,gestational diabetes, drug-induced diabetes, high blood glucose,metabolic syndrome, lipodystrophy syndrome, dyslipidemia, insulinresistance, leptin resistance, atherosclerosis, vascular disease,inflammatory disease, fibrotic disease, hypercholesterolemia,hypertriglyceridemia, non-alcoholic fatty liver disease, overweight, andobesity.

In one embodiment, the chimeric protein of the present invention orpharmaceutical composition thereof is administered with apharmaceutically-acceptable carrier.

The chimeric protein according to the present invention orpharmaceutical composition thereof can be administered orally,parenterally, subcutaneously, intravenously, intramuscularly,intraperitoneally, by intranasal instillation, by implantation, byintracavitary or intravesical instillation, intraocularly,intraarterially, intralesionally, transdermally, or by application tomucous membranes. The most suitable route may depend on the conditionand disorder of the recipient. Formulations including chimeric proteinsaccording to the present invention may conveniently be presented in unitdosage form and may be prepared by any of the methods well known in theart of pharmacy.

Dosages and desired drug concentrations of pharmaceutical compositionsof the present invention may vary depending on the particular useenvisioned. The determination of the appropriate dosage or route ofadministration is well within the skill of an ordinary physician. Thoseskilled in the art can readily optimize pharmaceutically effectivedosages and administration regimens for therapeutic compositionscomprising the chimeric protein according to the present invention, asdetermined by good medical practice and the clinical condition of theindividual patient.

When in vivo administration of a chimeric protein of the presentinvention or is employed, normal dosage amounts may vary from, forexample, about 10 ng/kg to up to 100 mg/kg of mammal body weight or moreper day. In one embodiment, the dosage may be from about 1 μg/kg/day to10 mg/kg/day, depending upon the route of administration. In oneembodiment, the chimeric protein according to the present invention isadministered at a dose of about 0.1 to 10 mg/kg once or twice daily. Inone embodiment, the chimeric protein according to the present inventionis administered at a dose of about 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to5, 1 to 4, 1 to 3, or 1 to 2 mg/kg. In one embodiment, the dosage is thesame as that of a native FGF21 therapeutic. In one embodiment, thedosage is less than that of a native FGF21 therapeutic, but has the sameeffect as a higher dosage of a native FGF21 therapeutic. Guidance as toparticular dosages and methods of delivery of proteins is provided inthe literature; see, for example, U.S. Pat. Nos. 4,657,760; 5,206,344;or 5,225,212, which are hereby incorporated by reference in theirentirety. It is anticipated that different formulations will beeffective for different treatment compounds and different disorders,that administration targeting one organ or tissue, for example, maynecessitate delivery in a manner different from that to another organ ortissue.

Where sustained-release administration of a chimeric protein of thepresent invention is desired in a formulation with releasecharacteristics suitable for the treatment of any disease or disorderrequiring administration of the chimeric protein of the presentinvention, microencapsulation is contemplated. Microencapsulation ofrecombinant proteins for sustained release has been successfullyperformed with human growth hormone (rhGH), interferon-(rhIFN-),interleukin-2, and MN rgp120. Johnson et al., “Preparation andCharacterization of Poly(D,L-lactide-co-glycolide) Microspheres forControlled Release of Human Growth Hormone,” Nat. Med. 2:795-799 (1996);Yasuda, “Sustained Release Formulation of Interferon,” Biomed. Ther.27:1221-1223 (1993); Hora et al., “Controlled Release of Interleukin-2from Biodegradable Microspheres,” Nat. Biotechnol. 8:755-758 (1990);Cleland, “Design and Production of Single Immunization Vaccines UsingPolylactide Polyglycolide Microsphere Systems,” in VACCINE DESIGN: THESUBUNIT AND ADJUVANT APPROACH 439-462 (Powell and Newman, eds. 1995); WO97/03692; WO 96/40072; WO 96/07399; and U.S. Pat. No. 5,654,010, whichare hereby incorporated by reference in their entirety. Thesustained-release formulations of these proteins were developed usingpoly-lactic-coglycolic acid (PLGA) polymer due to its biocompatibilityand wide range of biodegradable properties. The degradation products ofPLGA, lactic and glycolic acids, can be cleared quickly within the humanbody. Moreover, the degradability of this polymer can be adjusted frommonths to years depending on its molecular weight and composition.Lewis, “Controlled release of bioactive agents from lactide/glycolidepolymer,” in: BIODEGRADABLE POLYMERS AS DRUG DELIVERY SYSTEMS 1-41 (M.Chasin and R. Langer eds. 1990), which is hereby incorporated byreference in its entirety.

The chimeric protein of the present invention or pharmaceuticalcomposition thereof may be administered as frequently as necessary inorder to obtain the desired therapeutic effect. Some patients mayrespond rapidly to a higher or lower dose and may find much weakermaintenance doses adequate. For other patients, it may be necessary tohave long-term treatments at the rate of 1 to 4 doses per day, inaccordance with the physiological requirements of each particularpatient. For other patients, it will be necessary to prescribe not morethan one or two doses per day.

In some embodiments, the chimeric protein of the present invention or apharmaceutical composition thereof is administered in a therapeuticallyeffective amount in combination with a therapeutically effective amountof a second agent. In one embodiment, the chimeric protein of thepresent invention or pharmaceutical composition thereof is administeredin conjunction with the second agent, i.e., the respective periods ofadministration are part of a single administrative regimen. In oneembodiment, the chimeric protein of the present invention orpharmaceutical composition thereof and the second agent are administeredconcurrently, i.e., the respective periods of administration overlapeach other. In one embodiment, the chimeric protein of the presentinvention or pharmaceutical composition thereof and the second agent areadministered non-concurrently, i.e., the respective periods ofadministration do not overlap each other. In one embodiment, thechimeric protein of the present invention or pharmaceutical compositionthereof and the second agent are administered sequentially, i.e., thechimeric protein of the present invention or pharmaceutical compositionthereof is administered prior to and/or after the administration of thesecond agent. In one embodiment, the chimeric protein of the presentinvention or pharmaceutical composition thereof and the second agent areadministered simultaneously as separate compositions. In one embodiment,the chimeric protein of the present invention or pharmaceuticalcomposition thereof and the second agent are administered simultaneouslyas part of the same compositions.

In one embodiment, the second agent is an anti-inflammatory agent, ananti-fibrotic agent, an antihypertensive agent, an anti-diabetic agent,a triglyceride-lowering agent, and/or cholesterol-lowering drug such asa drug of the “statin” class. In one embodiment, the second agent isinsulin. In one embodiment, the insulin is rapid acting, short acting,regular acting, intermediate acting, or long acting insulin. In oneembodiment, the insulin is and/or comprises Humalog®, Lispro, Novolog®,Apidra®, Humulin®, Aspart, regular insulin, NPH, Lente, Ultralente,Lantus®, Glargine, Levemir®, or Detemir. In one embodiment, the secondagent is a statin. In one embodiment, the statin is and/or comprisesAtorvastatin (e.g., Lipitor® or Torvast®), Cerivastatin (e.g., Lipobay®or Baycol®), Fluvastatin (e.g., Lescol® or LescolXL®), Lovastatin (e.g.,Mevacor®, Altocor®, or Altoprev®) Mevastatin, Pitavastatin (e.g.,Livalo® or Pitava®), Pravastatin (e.g., Pravachol, Selektine, orLipostat®) Rosuvastatin (e.g., Crestor®), Simvastatin (e.g., Zocor® orLipex®), Vytorin®, Advicor®, Besylate Caduet® or Simcor®.

In one embodiment of the present invention, the chimeric proteinaccording to the present invention or the pharmaceutical compositionthereof is administered with an anti-inflammatory agent, an antifibroticagent, an antihypertensive agent, an antidiabetic agent, atriglyceride-lowering agent, and/or a cholesterol-lowering agent.

Another aspect of the present invention relates to a method of making achimeric FGF protein possessing enhanced endocrine activity. This methodinvolves introducing one or more modifications to a FGF protein, wherethe modification decreases the affinity of the FGF protein for heparinand/or heparan sulfate and coupling a C-terminal portion of FGF19 thatincludes a βKlotho co-receptor binding domain to the modified FGFprotein's C-terminus, whereby a chimeric FGF protein possessing enhancedendocrine activity is made.

Suitable C-terminal portions of FGF19 are described above. In oneembodiment, the C-terminal region from FGF19 is derived from a mammalianFGF19. In one embodiment, the C-terminal region derived from FGF19 isfrom a vertebrate FGF19.

In one embodiment, the chimeric FGF protein has greater binding affinityfor FGFR than native FGF19. In one embodiment the chimeric FGF proteinpossesses enhanced endocrine activity compared to the chimeric FGFprotein in the absence of the modification or the βKlotho co-receptorbinding domain. In one embodiment, the native endocrine FGF ligandhaving the βKlotho co-receptor binding domain is native FGF21. In oneembodiment, the FGFR is FGFR1c, FGFR2c, or FGFR4.

In one embodiment the chimeric FGF protein has greater stability than anative endocrine FGF ligand possessing the βKlotho co-receptor bindingdomain. In one embodiment, increasing the stability includes an increasein thermal stability of the protein as compared to either wild typeprotein or native endocrine FGF ligand. In one embodiment, increasingthe stability includes increasing the half-life of the protein in theblood circulation as compared to wild type protein or native endocrineFGF ligand.

In one embodiment, the method involves introducing one or moremodifications to the FGF protein, where the modification alters thereceptor-binding specificity of the FGF protein. In one embodiment, themethod involves introducing one or more modifications to the FGFprotein, where the modification alters the receptor-binding affinity ofthe FGF protein.

In one embodiment, the FGF is derived from a mammalian FGF. In oneembodiment, the FGF is derived from a vertebrate FGF. In one embodiment,the FGF protein is a paracrine FGF molecule. In one embodiment the FGFmolecule is FGF1 or FGF2. In one embodiment, the FGF protein is an FGFprotein that possesses intrinsically greater binding affinity for FGFreceptor than a native endocrine FGF ligand. In one embodiment, the FGFprotein is an FGF protein that possesses intrinsically greater thermalstability than a native endocrine FGF ligand. In one embodiment, themethod involves introducing one or more modifications to the FGFprotein, where the modification alters receptor-binding specificityand/or receptor-binding affinity of the FGF protein. In one embodiment,the method involves introducing one or more modifications to the FGFprotein, where the modification alters the stability of the FGF protein.For example, receptor-binding specificity of FGF1, which by nature bindsto all the seven principal FGFRs, may be altered to, for example, reduceany risk for adverse effects (e.g., mitogenicity). Paracrine FGFs,portions of paracrine FGFs, and modifications thereto are describedabove.

In one embodiment, the chimeric FGF protein is effective to treatdiabetes, obesity, and/or metabolic syndrome.

Suitable methods of generating chimeric proteins according to thepresent invention include standard methods of synthesis known in theart, as described above.

Yet another aspect of the present invention relates to a method offacilitating fibroblast growth factor receptor (“FGFR”)-βKlothoco-receptor complex formation. This method involves providing a cellthat includes a βKlotho co-receptor and an FGFR and providing a chimericFGF protein. The chimeric FGF protein includes a C-terminal portion ofFGF19 and a portion of a paracrine FGF, where the portion of theparacrine FGF is modified to decrease binding affinity for heparinand/or heparan sulfate compared to the portion without the modification.This method also involves contacting the cell and the chimeric FGFprotein under conditions effective to cause FGFR-βKlotho co-receptorcomplex formation.

Suitable portions of the paracrine FGFs for use in accordance with thepresent invention are described above. Suitable modifications to theparacrine FGFs for use in accordance with the present invention are alsodescribed above. Suitable C-terminal portions from FGF19 are describedabove and throughout the present application.

In one embodiment according to the present invention, βKlotho ismammalian βKlotho. In one embodiment, βKlotho is human or mouse βKlotho.In one particular embodiment of the present invention, βKlotho is humanor mouse βKlotho including the amino acid sequence of SEQ ID NO: 341(i.e., GenBank Accession No. NP_783864, which is hereby incorporated byreference in its entirety) or SEQ ID NO: 342 (i.e., GenBank AccessionNo. NP_112457, which is hereby incorporated by reference in itsentirety), respectively, as follows:

SEQ ID NO: 341: 1MKPGCAAGSP GNEWIFFSTD EITTRYRNTM SNGGLQRSVI LSALILLRAV TGFSGDGRAI 61WSKNPNFTPV NESQLFLYDT FPKNFFWGIG TGALQVEGSW KKDGKGPSIW DHFIHTHLKN 121VSSTNGSSDS YIFLEKDLSA LDFIGVSFYQ FSISWPRLFP DGIVTVANAK GLQYYSTLLD 181ALVLRNIEPI VTLYHWDLPL ALQEKYGGWK NDTIIDIFND YATYCFQMFG DRVKYWITIH 241NPYLVAWHGY GTGMHAPGEK GNLAAVYTVG HNLIKAHSKV WHNYNTHFRP HQKGWLSITL 301GSHWIEPNRS ENTMDIFKCQ QSMVSVLGWF ANPIHGDGDY PEGMRKKLFS VLPIFSEAEK 361HEMRGTADFF AFSFGPNNFK PLNTMAKMGQ NVSLNLREAL NWIKLEYNNP RILIAENGWF 421TDSRVKTEDT TAIYMMKNFL SQVLQAIRLD EIRVFGYTAW SLLDGFEWQD AYTIRRGLFY 481VDFNSKQKER KPKSSAHYYK QIIRENGFSL KESTPDVQGQ FPCDFSWGVT ESVLKPESVA 541SSPQFSDPHL YVWNATGNRL LHRVEGVRLK TRPAQCTDFV NIKKQLEMLA RMKVTHYRFA 601LDWASVLPTG NLSAVNRQAL RYYRCVVSEG LKLGISAMVT LYYPTHAHLG LPEPLLHADG 661WLNPSTAEAF QAYAGLCFQE LGDLVKLWIT INEPNRLSDI YNRSGNDTYG AAHNLLVAHA 721LAWRLYDRQF RPSQRGAVSL SLHADWAEPA NPYADSHWRA AERFLQFEIA WFAEPLFKTG 781DYPAAMREYI ASKHRRGLSS SALPRLTEAE RRLLKGTVDF CALNHFTTRF VMHEQLAGSR 841YDSDRDIQFL QDITRLSSPT RLAVIPWGVR KLLRWVRRNY GDMDIYITAS GIDDQALEDD 901RLRKYYLGKY LQEVLKAYLI DKVRIKGYYA FKLAEEKSKP RFGFFTSDFK AKSSIQFYNK 961VISSRGFPFE NSSSRCSQTQ ENTECTVCLF LVQKKPLIFL GCCFFSTLVL LLSIAIFQRQ 1021KRRKFWKAKN LQHIPLKKGK RVVS SEQ ID NO: 342: 1MKTGCAAGSP GNEWIFFSSD ERNTRSRKTM SNRALQRSAV LSAFVLLRAV TGFSGDGKAI 61WDKKQYVSPV NPSQLFLYDT FPKNFSWGVG TGAFQVEGSW KTDGRGPSIW DRYVYSHLRG 121VNGTDRSTDS YIFLEKDLLA LDFLGVSFYQ FSISWPRLFP NGTVAAVNAQ GLRYYRALLD 181SLVLRNIEPI VTLYHWDLPL TLQEEYGGWK NATMIDLFND YATYCFQTFG DRVKYWITIH 241NPYLVAWHGF GTGMHAPGEK GNLTAVYTVG HNLIKAHSKV WHNYDKNFRP HQKGWLSITL 301GSHWIEPNRT DNMEDVINCQ HSMSSVLGWF ANPIHGDGDY PEFMKTGAMI PEFSEAEKEE 361VRGTADFFAF SFGPNNFRPS NTVVKMGQNV SLNLRQVLNW IKLEYDDPQI LISENGWFTD 421SYIKTEDTTA IYMMKNFLNQ VLQAIKFDEI RVFGYTAWTL LDGFEWQDAY TTRRGLFYVD 481FNSEQKERKP KSSAHYYKQI IQDNGFPLKE STPDMKGRFP CDFSWGVTES VLKPEFTVSS 541PQFTDPHLYV WNVTGNRLLY RVEGVRLKTR PSQCTDYVSI KKRVEMLAKM KVTHYQFALD 601WTSILPTGNL SKVNRQVLRY YRCVVSEGLK LGVFPMVTLY HPTHSHLGLP LPLLSSGGWL 661NMNTAKAFQD YAELCFRELG DLVKLWITIN EPNRLSDMYN RTSNDTYRAA HNLMIAHAQV 721WHLYDRQYRP VQHGAVSLSL HCDWAEPANP FVDSHWKAAE RFLQFEIAWF ADPLFKTGDY 781PSVMKEYIAS KNQRGLSSSV LPRFTAKESR LVKGTVDFYA LNHFTTRFVI HKQLNTNRSV 841ADRDVQFLQD ITRLSSPSRL AVTPWGVRKL LAWIRRNYRD RDIYITANGI DDLALEDDQI 901RKYYLEKYVQ EALKAYLIDK VKIKGYYAFK LTEEKSKPRF GFFTSDFRAK SSVQFYSKLI 961SSSGLPAENR SPACGQPAED TDCTICSFLV EKKPLIFFGC CFISTLAVLL SITVFHHQKR 1021RKFQKARNLQ NIPLKKGHSR VFS

In one particular embodiment of the present invention, βKlotho is humanor mouse βKlotho encoded by a nucleotide sequence including thenucleotide sequences of SEQ ID NO: 343 (GenBank Accession No. NM_175737,which is hereby incorporated by reference in its entirety) and SEQ IDNO: 344 (GenBank Accession No. NM_031180, which is hereby incorporatedby reference in its entirety), as follows:

SEQ ID NO: 343 (Human βKlotho gene coding sequence): 98       ATG AAGCCAGGCT GTGCGGCAGG ATCTCCAGGG AATGAATGGA TTTTCTTCAG 151CACTGATGAA ATAACCACAC GCTATAGGAA TACAATGTCC AACGGGGGAT TGCAAAGATC 211TGTCATCCTG TCAGCACTTA TTCTGCTACG AGCTGTTACT GGATTCTCTG GAGATGGAAG 271AGCTATATGG TCTAAAAATC CTAATTTTAC TCCGGTAAAT GAAAGTCAGC TGTTTCTCTA 331TGACACTTTC CCTAAAAACT TTTTCTGGGG TATTGGGACT GGAGCATTGC AAGTGGAAGG 391GAGTTGGAAG AAGGATGGAA AAGGACCTTC TATATGGGAT CATTTCATCC ACACACACCT 451TAAAAATGTC AGCAGCACGA ATGGTTCCAG TGACAGTTAT ATTTTTCTGG AAAAAGACTT 511ATCAGCCCTG GATTTTATAG GAGTTTCTTT TTATCAATTT TCAATTTCCT GGCCAAGGCT 571TTTCCCCGAT GGAATAGTAA CAGTTGCCAA CGCAAAAGGT CTGCAGTACT ACAGTACTCT 631TCTGGACGCT CTAGTGCTTA GAAACATTGA ACCTATAGTT ACTTTATACC ACTGGGATTT 691GCCTTTGGCA CTACAAGAAA AATATGGGGG GTGGAAAAAT GATACCATAA TAGATATCTT 751CAATGACTAT GCCACATACT GTTTCCAGAT GTTTGGGGAC CGTGTCAAAT ATTGGATTAC 811AATTCACAAC CCATATCTAG TGGCTTGGCA TGGGTATGGG ACAGGTATGC ATGCCCCTGG 871AGAGAAGGGA AATTTAGCAG CTGTCTACAC TGTGGGACAC AACTTGATCA AGGCTCACTC 931GAAAGTTTGG CATAACTACA ACACACATTT CCGCCCACAT CAGAAGGGTT GGTTATCGAT 991CACGTTGGGA TCTCATTGGA TCGAGCCAAA CCGGTCGGAA AACACGATGG ATATATTCAA 1051ATGTCAACAA TCCATGGTTT CTGTGCTTGG ATGGTTTGCC AACCCTATCC ATGGGGATGG 1111CGACTATCCA GAGGGGATGA GAAAGAAGTT GTTCTCCGTT CTACCCATTT TCTCTGAAGC 1171AGAGAAGCAT GAGATGAGAG GCACAGCTGA TTTCTTTGCC TTTTCTTTTG GACCCAACAA 1231CTTCAAGCCC CTAAACACCA TGGCTAAAAT GGGACAAAAT GTTTCACTTA ATTTAAGAGA 1291AGCGCTGAAC TGGATTAAAC TGGAATACAA CAACCCTCGA ATCTTGATTG CTGAGAATGG 1351CTGGTTCACA GACAGTCGTG TGAAAACAGA AGACACCACG GCCATCTACA TGATGAAGAA 1411TTTCCTCAGC CAGGTGCTTC AAGCAATAAG GTTAGATGAA ATACGAGTGT TTGGTTATAC 1471TGCCTGGTCT CTCCTGGATG GCTTTGAATG GCAGGATGCT TACACCATCC GCCGAGGATT 1531ATTTTATGTG GATTTTAACA GTAAACAGAA AGAGCGGAAA CCTAAGTCTT CAGCACACTA 1591CTACAAACAG ATCATACGAG AAAATGGTTT TTCTTTAAAA GAGTCCACGC CAGATGTGCA 1651GGGCCAGTTT CCCTGTGACT TCTCCTGGGG TGTCACTGAA TCTGTTCTTA AGCCCGAGTC 1711TGTGGCTTCG TCCCCACAGT TCAGCGATCC TCATCTGTAC GTGTGGAACG CCACTGGCAA 1771CAGACTGTTG CACCGAGTGG AAGGGGTGAG GCTGAAAACA CGACCCGCTC AATGCACAGA 1831TTTTGTAAAC ATCAAAAAAC AACTTGAGAT GTTGGCAAGA ATGAAAGTCA CCCACTACCG 1891GTTTGCTCTG GATTGGGCCT CGGTCCTTCC CACTGGCAAC CTGTCCGCGG TGAACCGACA 1951GGCCCTGAGG TACTACAGGT GCGTGGTCAG TGAGGGGCTG AAGCTTGGCA TCTCCGCGAT 2011GGTCACCCTG TATTATCCGA CCCACGCCCA CCTAGGCCTC CCCGAGCCTC TGTTGCATGC 2071CGACGGGTGG CTGAACCCAT CGACGGCCGA GGCCTTCCAG GCCTACGCTG GGCTGTGCTT 2131CCAGGAGCTG GGGGACCTGG TGAAGCTCTG GATCACCATC AACGAGCCTA ACCGGCTAAG 2191TGACATCTAC AACCGCTCTG GCAACGACAC CTACGGGGCG GCGCACAACC TGCTGGTGGC 2251CCACGCCCTG GCCTGGCGCC TCTACGACCG GCAGTTCAGG CCCTCACAGC GCGGGGCCGT 2311GTCGCTGTCG CTGCACGCGG ACTGGGCGGA ACCCGCCAAC CCCTATGCTG ACTCGCACTG 2371GAGGGCGGCC GAGCGCTTCC TGCAGTTCGA GATCGCCTGG TTCGCCGAGC CGCTCTTCAA 2431GACCGGGGAC TACCCCGCGG CCATGAGGGA ATACATTGCC TCCAAGCACC GACGGGGGCT 2491TTCCAGCTCG GCCCTGCCGC GCCTCACCGA GGCCGAAAGG AGGCTGCTCA AGGGCACGGT 2551CGACTTCTGC GCGCTCAACC ACTTCACCAC TAGGTTCGTG ATGCACGAGC AGCTGGCCGG 2611CAGCCGCTAC GACTCGGACA GGGACATCCA GTTTCTGCAG GACATCACCC GCCTGAGCTC 2671CCCCACGCGC CTGGCTGTGA TTCCCTGGGG GGTGCGCAAG CTGCTGCGGT GGGTCCGGAG 2731GAACTACGGC GACATGGACA TTTACATCAC CGCCAGTGGC ATCGACGACC AGGCTCTGGA 2791GGATGACCGG CTCCGGAAGT ACTACCTAGG GAAGTACCTT CAGGAGGTGC TGAAAGCATA 2851CCTGATTGAT AAAGTCAGAA TCAAAGGCTA TTATGCATTC AAACTGGCTG AAGAGAAATC 2911TAAACCCAGA TTTGGATTCT TCACATCTGA TTTTAAAGCT AAATCCTCAA TACAATTTTA 2971CAACAAAGTG ATCAGCAGCA GGGGCTTCCC TTTTGAGAAC AGTAGTTCTA GATGCAGTCA 3031GACCCAAGAA AATACAGAGT GCACTGTCTG CTTATTCCTT GTGCAGAAGA AACCACTGAT 3091ATTCCTGGGT TGTTGCTTCT TCTCCACCCT GGTTCTACTC TTATCAATTG CCATTTTTCA 3151AAGGCAGAAG AGAAGAAAGT TTTGGAAAGC AAAAAACTTA CAACACATAC CATTAAAGAA 3211AGGCAAGAGA GTTGTTAGCT AASEQ ID NO: 344 (House mouse βKlotho gene coding sequence): 2ATGAAGACA GGCTGTGCAG CAGGGTCTCC GGGGAATGAA TGGATTTTCT TCAGCTCTGA 61TGAAAGAAAC ACACGCTCTA GGAAAACAAT GTCCAACAGG GCACTGCAAA GATCTGCCGT 121GCTGTCTGCG TTTGTTCTGC TGCGAGCTGT TACCGGCTTC TCCGGAGACG GGAAAGCAAT 181ATGGGATAAA AAACAGTACG TGAGTCCGGT AAACCCAAGT CAGCTGTTCC TCTATGACAC 241TTTCCCTAAA AACTTTTCCT GGGGCGTTGG GACCGGAGCA TTTCAAGTGG AAGGGAGTTG 301GAAGACAGAT GGAAGAGGAC CCTCGATCTG GGATCGGTAC GTCTACTCAC ACCTGAGAGG 361TGTCAACGGC ACAGACAGAT CCACTGACAG TTACATCTTT CTGGAAAAAG ACTTGTTGGC 421TCTGGATTTT TTAGGAGTTT CTTTTTATCA GTTCTCAATC TCCTGGCCAC GGTTGTTTCC 481CAATGGAACA GTAGCAGCAG TGAATGCGCA AGGTCTCCGG TACTACCGTG CACTTCTGGA 541CTCGCTGGTA CTTAGGAATA TCGAGCCCAT TGTTACCTTG TACCATTGGG ATTTGCCTCT 601GACGCTCCAG GAAGAATATG GGGGCTGGAA AAATGCAACT ATGATAGATC TCTTCAACGA 661CTATGCCACA TACTGCTTCC AGACCTTTGG AGACCGTGTC AAATATTGGA TTACAATTCA 721CAACCCTTAC CTTGTTGCTT GGCATGGGTT TGGCACAGGT ATGCATGCAC CAGGAGAGAA 781GGGAAATTTA ACAGCTGTCT ACACTGTGGG ACACAACCTG ATCAAGGCAC ATTCGAAAGT 841GTGGCATAAC TACGACAAAA ACTTCCGCCC TCATCAGAAG GGTTGGCTCT CCATCACCTT 901GGGGTCCCAT TGGATAGAGC CAAACAGAAC AGACAACATG GAGGACGTGA TCAACTGCCA 961GCACTCCATG TCCTCTGTGC TTGGATGGTT CGCCAACCCC ATCCACGGGG ACGGCGACTA 1021CCCTGAGTTC ATGAAGACGG GCGCCATGAT CCCCGAGTTC TCTGAGGCAG AGAAGGAGGA 1081GGTGAGGGGC ACGGCTGATT TCTTTGCCTT TTCCTTCGGG CCCAACAACT TCAGGCCCTC 1141AAACACCGTG GTGAAAATGG GACAAAATGT ATCACTCAAC TTAAGGCAGG TGCTGAACTG 1201GATTAAACTG GAATACGATG ACCCTCAAAT CTTGATTTCG GAGAACGGCT GGTTCACAGA 1261TAGCTATATA AAGACAGAGG ACACCACGGC CATCTACATG ATGAAGAATT TCCTAAACCA 1321GGTTCTTCAA GCAATAAAAT TTGATGAAAT CCGCGTGTTT GGTTATACGG CCTGGACTCT 1381CCTGGATGGC TTTGAGTGGC AGGATGCCTA TACGACCCGA CGAGGGCTGT TTTATGTGGA 1441CTTTAACAGT GAGCAGAAAG AGAGGAAACC CAAGTCCTCG GCTCATTACT ACAAGCAGAT 1501CATACAAGAC AACGGCTTCC CTTTGAAAGA GTCCACGCCA GACATGAAGG GTCGGTTCCC 1561CTGTGATTTC TCTTGGGGAG TCACTGAGTC TGTTCTTAAG CCCGAGTTTA CGGTCTCCTC 1621CCCGCAGTTT ACCGATCCTC ACCTGTATGT GTGGAATGTC ACTGGCAACA GATTGCTCTA 1681CCGAGTGGAA GGGGTAAGGC TGAAAACAAG ACCATCCCAG TGCACAGATT ATGTGAGCAT 1741CAAAAAACGA GTTGAAATGT TGGCAAAAAT GAAAGTCACC CACTACCAGT TTGCTCTGGA 1801CTGGACCTCT ATCCTTCCCA CTGGCAATCT GTCCAAAGTT AACAGACAAG TGTTAAGGTA 1861CTATAGGTGT GTGGTGAGCG AAGGACTGAA GCTGGGCGTC TTCCCCATGG TGACGTTGTA 1921CCACCCAACC CACTCCCATC TCGGCCTCCC CCTGCCACTT CTGAGCAGTG GGGGGTGGCT 1981AAACATGAAC ACAGCCAAGG CCTTCCAGGA CTACGCTGAG CTGTGCTTCC GGGAGTTGGG 2041GGACTTGGTG AAGCTCTGGA TCACCATCAA TGAGCCTAAC AGGCTGAGTG ACATGTACAA 2101CCGCACGAGT AATGACACCT ACCGTGCAGC CCACAACCTG ATGATCGCCC ATGCCCAGGT 2161CTGGCACCTC TATGATAGGC AGTATAGGCC GGTCCAGCAT GGGGCTGTGT CGCTGTCCTT 2221ACATTGCGAC TGGGCAGAAC CTGCCAACCC CTTTGTGGAT TCACACTGGA AGGCAGCCGA 2281GCGCTTCCTC CAGTTTGAGA TCGCCTGGTT TGCAGATCCG CTCTTCAAGA CTGGCGACTA 2341TCCATCGGTT ATGAAGGAAT ACATCGCCTC CAAGAACCAG CGAGGGCTGT CTAGCTCAGT 2401CCTGCCGCGC TTCACCGCGA AGGAGAGCAG GCTGGTGAAG GGTACCGTCG ACTTCTACGC 2461ACTGAACCAC TTCACTACGA GGTTCGTGAT ACACAAGCAG CTGAACACCA ACCGCTCAGT 2521TGCAGACAGG GACGTCCAGT TCCTGCAGGA CATCACCCGC CTAAGCTCGC CCAGCCGCCT 2581GGCTGTAACA CCCTGGGGAG TGCGCAAGCT CCTTGCGTGG ATCCGGAGGA ACTACAGAGA 2641CAGGGATATC TACATCACAG CCAATGGCAT CGATGACCTG GCTCTAGAGG ATGATCAGAT 2701CCGAAAGTAC TACTTGGAGA AGTATGTCCA GGAGGCTCTG AAAGCATATC TCATTGACAA 2761GGTCAAAATC AAAGGCTACT ATGCATTCAA ACTGACTGAA GAGAAATCTA AGCCTAGATT 2821TGGATTTTTC ACCTCTGACT TCAGAGCTAA GTCCTCTGTC CAGTTTTACA GCAAGCTGAT 2881CAGCAGCAGT GGCCTCCCCG CTGAGAACAG AAGTCCTGCG TGTGGTCAGC CTGCGGAAGA 2941CACAGACTGC ACCATTTGCT CATTTCTCGT GGAGAAGAAA CCACTCATCT TCTTCGGTTG 3001CTGCTTCATC TCCACTCTGG CTGTACTGCT ATCCATCACC GTTTTTCATC ATCAAAAGAG 3061AAGAAAATTC CAGAAAGCAA GGAACTTACA AAATATACCA TTGAAGAAAG GCCACAGCAG 3121AGTTTTCAGC TAA

In one embodiment, the FGFR is FGFR1c, FGFR2c, or FGFR4. In oneembodiment of the present invention, the FGF receptor is FGFR1creceptor. In one particular embodiment, the FGFR1c receptor is the humanFGFR1c receptor (GenBank Accession No. NP_075598, which is herebyincorporated by reference in its entirety). In another embodiment, theFGF receptor is FGFR2c receptor. In one particular embodiment, theFGFR2c receptor is the human FGFR2c receptor (GenBank Accession No.NP_000132, which is hereby incorporated by reference in its entirety).In another embodiment, the FGF receptor is FGFR4 receptor. In oneparticular embodiment, the FGFR4 receptor is the human FGFR4 receptor(GenBank Accession No. NP002002, which is hereby incorporated byreference in its entirety).

In one embodiment, the method of facilitating FGFR-βKlotho co-receptorcomplex formation is carried out in vitro. In one embodiment, the methodis carried out in an adipocyte. In another embodiment, the method iscarried out in a skeletal muscle cell, a pancreatic β cell, or ahepatocyte.

In one embodiment, the method of facilitating FGFR-βKlotho co-receptorcomplex formation is carried out in vivo. In one embodiment, the methodis carried out in a mammal. In one particular embodiment, the mammal isa mouse. In one embodiment, the mouse is an ob/ob or db/db mouse.

Yet a further aspect of the present invention relates to a method ofscreening for agents capable of facilitating FGFR-βKlotho complexformation in the treatment of a disorder. This method involves providinga chimeric FGF that includes an N-terminus coupled to a C-terminus,where the N-terminus includes a portion of a paracrine FGF and theC-terminus includes a C-terminal portion of FGF19. The portion of theparacrine FGF is modified to decrease binding affinity for heparinand/or heparan sulfate compared to the portion without the modification.The portion of the paracrine FGF may also be modified to alterreceptor-binding specificity and/or receptor-binding affinity comparedto the portion without the modification. This method also involvesproviding a binary βKlotho-FGFR complex and providing one or morecandidate agents. This method further involves combining the chimericFGF, the binary βKlotho-FGFR complex, and the one or more candidateagents under conditions permitting the formation of a ternary complexbetween the chimeric FGF and the binary βKlotho-FGFR complex in theabsence of the one or more candidate agents. This method also involvesidentifying the one or more candidate agents that decrease ternarycomplex formation between the chimeric FGF and the binary βKlotho-FGFRcomplex compared to the ternary complex formation in the absence of theone or more candidate agents as suitable for treating the disorder.

The portion of the paracrine FGF may also be modified to alterreceptor-binding specificity and/or reduce receptor-binding affinitycompared to the portion without the modification.

Suitable chimeric proteins for use in accordance with this aspect of thepresent invention are described above and throughout the presentapplication. Suitable paracrine FGFs, as well as suitable modificationsto decrease binding affinity for heparin and/or heparan sulfate, toalter receptor-binding specificity and/or receptor-binding affinitycompared to the portion without the modification, are also describedabove.

In one embodiment, the modulation is a competitive interaction betweenthe chimeric FGF molecule and the one or more candidate agents forbinding to the binary βKlotho-FGFR complex.

In one embodiment, the FGFR is FGFR1c, FGFR2c, or FGFR4.

In one embodiment, the disorder is a selected from diabetes, obesity,and metabolic syndrome. In one embodiment, the disorder is diabetesselected from type II diabetes, gestational diabetes, or drug-induceddiabetes. In one embodiment, the disorder is type I diabetes. In oneembodiment, the disorder is obesity. In one embodiment, the disorder ismetabolic syndrome.

In one embodiment of the screening aspects of the present invention, aplurality of compounds or agents is tested. Candidate agents may includesmall molecule compounds or larger molecules (e.g., proteins orfragments thereof). In one embodiment, the candidate compounds arebiomolecules. In one embodiment, the biomolecules are proteins. In oneembodiment, the biomolecules are peptides. In one embodiment, thecandidates are peptides or peptide mimetics having similar structuralfeatures to native FGF ligand. In one embodiment, the candidate agent isa second chimeric FGF molecule. In one particular embodiment, thepeptides are synthetic peptides. In one embodiment, the compounds aresmall organic molecules.

In one embodiment of the screening aspects of the present invention, themethod is carried out using a cell-based assay. In one embodiment, theidentifying is carried out using a cell-based assay.

In one embodiment of the screening aspects of the present invention, themethod is carried out using a binding assay. In one embodiment, thebinding assay is a direct binding assay. In one embodiment, the bindingassay is a competition-binding assay. In one embodiment, the modulationstabilizes the ternary complex between the chimeric FGF molecule and thebinary βKlotho-FGFR complex. In one embodiment, the stabilization iscompared to the native ternary complex.

In one embodiment, the modulation is an allosteric or kineticmodulation. In one embodiment, the allosteric or kinetic modulation iscompared to the native ternary complex. Such stabilization or allostericor kinetic modulation can be measured using methods known in the art(e.g., by use of surface plasmon resonance (SPR) spectroscopyexperiments as described in the Examples infra).

In one embodiment, the binding assay is carried out using surfaceplasmon resonance spectroscopy. In one embodiment, the identifying iscarried out using a binding assay. In one embodiment, the identifying iscarried out using surface plasmon resonance spectroscopy.

In one embodiment of the screening aspects of the present invention, thecell-based assay is carried out with adipocytes. In one embodiment, thecell-based assay is carried out with skeletal muscle cells. In oneembodiment, the cell-based assay is carried out with pancreatic β cells.In one embodiment, the cell-based assay is carried out with hepatocytes.In one embodiment, stimulation of glucose uptake is the assay readout.In one embodiment, induction of glucose transporter 1 gene expression isthe assay readout. In one embodiment, a dose-response curve is generatedfor the stimulation of glucose uptake by a candidate compound todetermine potency and efficacy of the candidate compound. In oneembodiment, a dose-response curve is generated for the induction ofglucose transporter 1 gene expression by a candidate compound todetermine potency and efficacy of the candidate compound. For example,if the dose-response curve is shifted to the left compared to thatobtained for the chimeric FGF protein, the candidate compound hasgreater potency than the chimeric FGF protein and/or native FGF19. Inone embodiment, an IC₅₀ value is derived from the dose-response curve ofa candidate compound to determine potency of the candidate compound. AnIC₅₀ value smaller than that obtained for the chimeric FGF proteinidentifies a candidate compound as more potent than the chimeric FGFprotein and/or native FGF19.

In one embodiment of the screening aspects of the present invention, thecell-based assay is carried out with mammalian cells ectopicallyexpressing βKlotho. In one particular embodiment, the cells are HEK293cells. In one embodiment, activation of FGF receptor is the assayreadout. In one embodiment, tyrosine phosphorylation of an FGF receptorsubstrate is used as readout for FGF receptor activation. In oneparticular embodiment, the FGF receptor substrate is FGF receptorsubstrate 2α. In one embodiment, activation of downstream mediators ofFGF signaling is used as readout for (or an indicator of) FGF receptoractivation. In one particular embodiment, the downstream mediator of FGFsignaling is 44/42 mitogen-activated protein kinase. In one embodiment,the downstream mediator of FGF signaling is a transcription factor. Inone particular embodiment, the transcription factor is early growthresponse 1. In one embodiment, a dose-response curve is generated forβKlotho-dependent activation of FGF receptor by a candidate compound todetermine potency and efficacy of the candidate compound. For example,if the dose-response curve is shifted to the left compared to thatobtained for the chimeric FGF protein, the candidate compound is morepotent than the chimeric FGF protein and/or native FGF19. In oneembodiment, an IC₅₀ value is derived from the dose-response curve of acandidate compound to determine potency of the candidate compound. AnIC₅₀ value smaller than that obtained for the chimeric FGF proteinidentifies a candidate compound as more potent than the chimeric FGFprotein and/or native FGF19.

In one embodiment of the screening aspects of the present invention, thesurface plasmon resonance spectroscopy-based assay is carried out usingthe chimeric FGF protein as ligand coupled to a biosensor chip. In oneembodiment, mixtures of βKlotho ectodomain with increasingconcentrations of a candidate compound are passed over a biosensor chipcontaining chimeric FGF protein. In one embodiment, mixtures of thebinary complex of FGFR ligand-binding domain and βKlotho ectodomain withincreasing concentrations of a candidate compound are passed over abiosensor chip containing chimeric FGF protein. In one particularembodiment, the FGFR ligand-binding domain is the FGFR1c ligand-bindingdomain. In one embodiment, an inhibition-binding curve is plotted for acandidate compound to determine potency of the candidate compound. Forexample, if the inhibition-binding curve is shifted to the left comparedto that obtained for the chimeric FGF protein, the candidate compoundhas greater potency than the chimeric FGF protein and/or native FGF19.In one embodiment, an IC₅₀ value is derived from the inhibition-bindingcurve of a candidate compound to determine potency of the candidatecompound. An IC₅₀ value smaller than that obtained for containingchimeric FGF protein identifies a candidate compound as more potent thanthe chimeric FGF protein and/or native FGF19. In one embodiment, theinhibition constant K_(i) is determined for a candidate compound todetermine potency of the candidate compound. A K_(i) value smaller thanthat obtained for native FGF19 identifies a candidate compound as morepotent than the chimeric FGF protein and/or native FGF19.

In one embodiment of the screening aspects of the present invention, themethod is carried out in vivo. In one embodiment, the method is carriedout in a mammal. In one particular embodiment, the mammal is a mouse. Inone embodiment, the mammal has obesity, diabetes, or a related metabolicdisorder. In one embodiment, the ability of a candidate compound topotentiate the hypoglycemic effect of insulin is used as readout forFGF19-like metabolic activity. This involves fasting the mammal for aperiod of time prior to insulin injection and measuring fasting bloodglucose levels. The mammal is then injected with insulin alone orco-injected with insulin plus a candidate compound. Blood glucose levelsare measured at several time points after the injection. If a candidatecompound potentiates the hypoglycemic effect of insulin to a greaterdegree than the chimeric FGF protein and/or native FGF19 does, thecandidate compound exhibits enhanced efficacy. Likewise, if a candidatecompound potentiates the hypoglycemic effect of insulin to a similardegree than the chimeric FGF protein and/or native FGF19 does but at alower dose compared to that of the chimeric FGF protein and/or nativeFGF19 and/or for a longer period of time compared to the chimeric FGFprotein and/or native FGF19, the candidate compound has enhancedagonistic properties. In one embodiment, the ability of a candidatecompound to elicit a hypoglycemic effect in a mammal with diabetes,obesity, or a related metabolic disorder is used as readout forFGF21-like metabolic activity. This involves injecting a mammalsuffering from diabetes, obesity, or a related metabolic disorder withthe candidate compound. Blood glucose levels are measured before theinjection and at several time points thereafter. If a candidate compoundhas a greater hypoglycemic effect than the chimeric FGF protein and/ornative FGF21 does, the candidate compound exhibits enhanced efficacy.Likewise, if a candidate compound shows a similar hypoglycemic effectthan the chimeric FGF protein and/or native FGF21 does but at a lowerdose compared to that of the chimeric FGF protein and/or native FGF21and/or for a longer period of time compared to the chimeric FGF proteinand/or native FGF21, the candidate compound has enhanced agonisticproperties.

EXAMPLES Example 1—Purification of FGF, FGFR, and Klotho Proteins

The N-terminally hexahistidine-tagged, mature form of human FGF19 (SEQID NO: 233) (R23 to K216), human FGF21 (SEQ ID NO: 332) (H29 to S209;FIG. 5A), and human FGF23 (Y25 to I251; FIG. 5A) was refolded in vitrofrom bacterial inclusion bodies, and purified by published protocols(Ibrahimi et al., Hum. Mol. Genet. 13:2313-2324 (2004); Plotnikov etal., Cell 101:413-424 (2000), which is hereby incorporated by referencein its entirety). The amino acid sequence of human FGF23 (SEQ ID NO:345)(GenBank accession no. AAG09917, which is hereby incorporated byreference in its entirety) is as follows:

1 MLGARLRLWV CALCSVCSMS VLRAYPNASP LLGSSWGGLI HLYTATARNS YHLQIHKNGH 61VDGAPHQTIY SALMIRSEDA GFVVITGVMS RRYLCMDFRG NIFGSHYFDP ENCRFQHQTL 121ENGYDVYHSP QYHFLVSLGR AKRAFLPGMN PPPYSQFLSR RNEIPLIHFN TPIPRRHTRS 181AEDDSERDPL NVLKPRARMT PAPASCSQEL PSAEDNSPMA SDPLGVVRGG RVNTHAGGTG 241PEGCRPFAKF I

HS-binding site mutants of FGF19 (K149A) and FGF23 (R140A/R143A) werepurified from bacterial inclusion bodies by similar protocols as thewild-type proteins. In order to minimize proteolysis of FGF23 wild-typeand mutant proteins, arginine residues 176 and 179 of the proteolyticcleavage site ¹⁷⁶RXXR¹⁷⁹ were replaced with glutamine as it occurs inthe phosphate wasting disorder “autosomal dominant hypophosphatemicrickets” (ADHR) (White et al., Nat. Genet. 26:345-348 (2000); White etal., Kidney Int. 60:2079-2086 (2001), which are hereby incorporated byreference in their entirety). Human FGF1 (M1 to D155; FIG. 6),N-terminally truncated human FGF1 (K25 to D155, termed FGF1^(ΔNT); FIG.6), human FGF2 (M1 to S155; FIG. 5A), and human FGF homologous factor 1B(FHF1B; M1 to T181) were purified by published protocols (Plotnikov etal., Cell 101:413-424 (2000); Olsen et al., J. Biol. Chem.278:34226-34236 (2003), which are hereby incorporated by reference intheir entirety).

Chimeras composed of the core domain of FGF2 (M1 to M151) and theC-terminal region of either FGF21 (P168 to S209) or FGF23 (R161 to I251)(termed FGF2^(WTcore)-FGF21^(C-tail) and FGF2^(WTcore)-FGF23^(C-tail),respectively; FIG. 5A) were purified by the same protocol as that fornative FGF2 (Plotnikov et al., Cell 101:413-424 (2000), which is herebyincorporated by reference in its entirety). Analogous chimerascontaining three mutations in the HS-binding site of the FGF2 core(K128D/R129Q/K134V) (termed FGF2a^(ΔHBScore)-FGF21^(C-tail) andFGF2^(ΔHBScore)-FGF23^(C-tail), respectively, FIG. 5A) were purifiedfrom the soluble bacterial cell lysate fraction by ion-exchange andsize-exclusion chromatographies. In order to minimize proteolysis of thechimeras containing the C-terminal sequence from R161 to I251 of FGF23,arginine residues 176 and 179 of the proteolytic cleavage site¹⁷⁶RXXR¹⁷⁹ located within this sequence were replaced with glutamine asit occurs in ADHR (White et al., Nat. Genet. 26:345-348 (2000); White etal., Kidney Int. 60:2079-2086 (2001), which are hereby incorporated byreference in their entirety). In addition, in order to preventdisulfide-mediated dimerization of FGF2 and chimeric FGF2 proteins,cysteine residues 78 and 96 were mutated to serine. An HS-binding sitemutant of FGF1 (K127D/K128Q/K133V) (termed FGF1^(ΔHBScore); FIG. 6) andchimeras composed of the core domain of the HS-binding site mutant ofFGF1 (M1 to L150, K127D/K128Q/K133V) and the C-terminal region of eitherFGF19 (L169 to K216) or FGF21 (P168 to S209) (termedFGF1^(ΔHBScore)-FGF19^(C-tail) and FGF1^(ΔHBScore)-FGF21^(C-tail),respectively; FIG. 6) were purified from the soluble bacterial celllysate fraction by ion-exchange and size-exclusion chromatographies. TheN-terminally hexahistidine-tagged C-terminal tail peptide of FGF23 (S180to I251, termed FGF23^(C-tail)) was purified by a published protocol(Goetz et al., Proc. Nat'l. Acad. Sci. U.S.A 107:407-412 (2010), whichis hereby incorporated by reference in its entirety). The ligand-bindingdomain of human FGFR1c (D142 to R365) was refolded in vitro frombacterial inclusion bodies, and purified by published protocols(Ibrahimi et al., Hum. Mol. Genet. 13:2313-2324 (2004); Plotnikov etal., Cell 101:413-424 (2000), which are hereby incorporated by referencein their entirety). The ectodomain of murine αKlotho (A35 to K982) andthe ectodomain of murine βKlotho (F53 to L995) were expressed in HEK293cells as fusion proteins with a C-terminal FLAG tag (Kurosu et al., J.Biol. Chem. 281:6120-6123 (2006); Kurosu et al., Science 309:1829-1833(2005), which are hereby incorporated by reference in their entirety).The binary complex of FGFR1c ligand-binding domain with αKlothoectodomain (referred to as αKlotho-FGFR1c complex) was prepared by apublished protocol (Goetz et al., Proc. Nat'l. Acad. Sci. U.S.A107:407-412 (2010), which is hereby incorporated by reference in itsentirety). The binary complex of FGFR1c ligand-binding domain withβKlotho ectodomain (referred to as βKlotho-FGFR1c complex) was preparedin the same fashion as the αKlotho-FGFR1c complex.

Example 2—Analysis of FGF-Heparin and FGF-FGFR-α/βKlotho Interactions bySurface Plasmon Resonance Spectroscopy

Surface plasmon resonance (SPR) experiments were performed on a Biacore2000 instrument (Biacore AB), and the interactions were studied at 25°C. in HBS-EP buffer (10 mM HEPES-NaOH, pH 7.4, 150 mM NaCl, 3 mM EDTA,0.005% (v/v) polysorbate 20). To study endocrine FGF-heparininteractions, a heparin chip was prepared by immobilizing biotinylatedheparin (Sigma-Aldrich) on flow channels of a research-gradestreptavidin chip (Biacore AB). The coupling density was ˜5 fmol mm⁻² offlow channel. To measure binding of chimeric FGF2 proteins to heparin,biotinylated heparin was coupled to a streptavidin chip at anapproximately 4-fold lower density as judged based on the bindingresponses obtained for FGF1. To study FGF-FGFR-α/βKlotho interactions,FGF chips were prepared by covalent coupling of FGF proteins throughtheir free amino groups on flow channels of research grade CM5 chips(Biacore AB). Proteins were injected over a chip at a flow rate of 50 μlmin⁻¹, and at the end of each protein injection (180 and 300 s,respectively), HBS-EP buffer (50 μl min⁻¹) was flowed over the chip tomonitor dissociation for 180 or 240 s. The heparin chip surface wasregenerated by injecting 50 μl of 2.0 M NaCl in 10 mM sodium acetate, pH4.5. For FGF chips, regeneration was achieved by injecting 2.0 M NaCl in10 mM sodium/potassium phosphate, pH 6.5. To control for nonspecificbinding in experiments where an FGF ligand was immobilized on the chip,FHF1B, which shares structural similarity with FGFs but does not exhibitany FGFR binding (Olsen et al., J. Biol. Chem. 278:34226-34236 (2003),which is hereby incorporated by reference in its entirety), was coupledto the control flow channel of the chip (˜15-30 fmol mm²). Inexperiments where heparin was immobilized on the chip, the control flowchannel was left blank. The data were processed with BiaEvaluationsoftware (Biacore AB). For each protein injection over the heparin chip,the nonspecific responses from the control flow channel were subtractedfrom the responses recorded for the heparin flow channel. Similarly, foreach protein injection over a FGF chip, the nonspecific responses fromthe FHF1B control flow channel were subtracted from the responsesrecorded for the FGF flow channel. Where possible, equilibriumdissociation constants (K_(D)s) were calculated from fitted saturationbinding curves. Fitted binding curves were judged to be accurate basedon the distribution of the residuals (even and near zero) and χ² (<10%of R_(max)).

To examine whether the K149A mutation abrogates residual heparin bindingof FGF19, increasing concentrations of wild-type FGF19 were passed overa heparin chip. Thereafter, the FGF19^(K149A) mutant was injected overthe heparin chip at the highest concentration tested for the wild-typeligand. The effect of the R140A/R143A double mutation in the HS-bindingsite of FGF23 on residual heparin binding of FGF23 was examined in thesame fashion as was the effect of the HS-binding site mutation in FGF19.

To verify that the K128D/R129Q/K134V triple mutation in the HS-bindingsite of the FGF2 core domain diminishes heparin-binding affinity of theFGF2 core, increasing concentrations of FGF2^(ΔHBScore)-FGF21^(C-tail)and FGF2^(ΔHBScore)-FGF23^(C-tail) were passed over a heparin chip. As acontrol, binding of FGF2^(WTcore)-FGF21^(C-tail) andFGF2^(WTcore)-FGF23^(C-tail) to heparin was studied.

To examine whether the FGF2^(ΔHBScore)-FGF23^(C-tail) chimera cancompete with FGF23 for binding to the αKlotho-FGFR1c complex, FGF23 wasimmobilized on a chip (˜16 fmol mm⁻² of flow channel). Increasingconcentrations of FGF2^(ΔHBScore)-FGF23^(C-tail) were mixed with a fixedconcentration of αKlotho-FGFR1c complex in HBS-EP buffer, and themixtures were injected over the FGF23 chip. As controls, the bindingcompetition was carried out with FGF23 or FGF2 as the competitor insolution. As an additional specificity control, competition of theFGF2^(ΔHBScore)-FGF23^(C-tail) chimera with FGF21 for binding to theαKlotho-FGFR1c complex was studied. αKlotho-FGFR1c complex was mixedwith FGF2^(ΔHBScore)-FGF23^(C-tail) or FGF23 at a molar ratio of 1:10,and the mixture was injected over a chip containing immobilized FGF21(˜12 fmol mm⁻² of flow channel).

To test whether the FGF2^(ΔHBScore)-FGF21^(C-tail) chimera can competewith FGF21 for binding to the βKlotho-FGFR1c complex, increasingconcentrations of FGF2^(ΔHBScore)-FGF21^(C-tail) were mixed with a fixedconcentration of βKlotho-FGFR1c complex in HBS-EP buffer, and themixtures were passed over a chip containing immobilized FGF21 (˜19 fmolmm⁻² of flow channel). As controls, the binding competition was carriedout with FGF21 or FGF2 as the competitor in solution. As an additionalspecificity control, competition of the FGF2^(ΔHBScore)-FGF21^(C-tail)chimera with FGF23 for binding to the αKlotho-FGFR1c complex wasstudied. αKlotho-FGFR1c complex was mixed withFGF2^(ΔHBScore)-FGF21^(C-tail) or FGF21 at a molar ratio of 1:10, andthe mixture was injected over a chip containing immobilized FGF23 (˜12fmol mm⁻² of flow channel).

To measure binding of FGFR1c to each of the three endocrine FGFs,increasing concentrations of FGFR1c ligand-binding domain were injectedover a chip containing immobilized FGF19, FGF21, and FGF23 (˜30 fmolmm⁻² of flow channel). As a control, binding of FGFR1c to FGF2immobilized on a chip was studied. As additional controls, binding ofthe αKlotho-FGFR1c complex to FGF23 and binding of FGFR1c to theC-terminal tail peptide of FGF23 was measured.

Example 3—Analysis of Phosphorylation of FRS2α and 44/42 MAP Kinase inHepatoma and Epithelial Cell Lines

To examine whether the FGF19^(K149A) and FGF23^(R140A/R143A) mutants canactivate FGFR in a α/βKlotho-dependent fashion, induction of tyrosinephosphorylation of FGFR substrate 2α (FRS2α) and downstream activationof MAP kinase cascade was used as readout for FGFR activation.Subconfluent cells of the H4IIE rat hepatoma cell line, whichendogenously expresses βKlotho (Kurosu et al., J. Biol. Chem.282:26687-26695 (2007), which is hereby incorporated by reference in itsentirety), were serum starved for 16 h and then stimulated for 10 minwith the FGF19^(K149A) mutant or wild-type FGF19 (0.2 ng ml⁻¹ to 2.0 μgml⁻¹). Similarly, subconfluent cells of a HEK293 cell line ectopicallyexpressing the transmembrane isoform of murine αKlotho (Kurosu et al.,J. Biol. Chem. 281:6120-6123 (2006), which is hereby incorporated byreference in its entirety) were treated with the FGF23^(R140A/R143A)mutant or wild-type FGF23 (0.1 to 100 ng ml⁻¹). After stimulation, thecells were lysed (Kurosu et al., Science 309:1829-1833 (2005), which ishereby incorporated by reference in its entirety), and cellular proteinswere resolved on SDS-polyacrylamide gels and transferred tonitrocellulose membranes. The protein blots were probed with antibodiesto phosphorylated FRS2α, phosphorylated 44/42 MAP kinase, total(phosphorylated and nonphosphorylated) 44/42 MAP kinase, and αKlotho.Except for the anti-αKlotho antibody (KM2119) (Kato et al., Biochem.Biophys. Res. Commun. 267:597-602 (2000), which is hereby incorporatedby reference in its entirety), all antibodies were from Cell SignalingTechnology.

Example 4—Analysis of Egr1 Protein Expression in an Epithelial Cell Line

To examine whether the FGF2^(ΔHBScore)-FGF21^(C-tail) andFGF2^(ΔHBScore)-FGF23^(C-tail) chimeras can activate FGFR in aHS-dependent fashion, induction of protein expression of thetranscription factor early growth response 1 (Egr1), a known downstreammediator of FGF signaling, was used as readout for FGFR activation.HEK293 cells were serum starved overnight and then stimulated for 90 minwith FGF2^(ΔHBScore)FGF21^(C-tail) or FGF2^(ΔHBScore)-FGF23^(C-tail)(0.1 and 0.3 nM). Cell stimulation with FGF2^(WTcore)-FGF21^(C-tail),FGF2^(WTcore)-FGF23^(C-tail), FGF21, and FGF23 served as controls. Totest whether the FGF2^(ΔHBScore) FGF21^(C-tail) chimera can activateFGFR in a βKlotho-dependent fashion, HEK293 cells transfected withmurine βKlotho were serum starved overnight and then stimulated for 90min with FGF2^(ΔHBScore)-FGF21^(C-tail) or FGF21 (3 to 300 ng ml⁻¹).After stimulation, the cells were lysed (Kurosu et al., Science309:1829-1833 (2005), which is hereby incorporated by reference in itsentirety), and cellular proteins were resolved on SDS-polyacrylamidegels and transferred to nitrocellulose membranes. The protein blots wereprobed with antibodies to Egr1 and glyceraldehyde 3-phosphatedehydrogenase (GAPDH). The anti-Egr1 antibody was from Cell SignalingTechnology and the anti-GAPDH antibody was from Abcam.

Example 5—Analysis of CYP7A1 and CYP8B1 mRNA Expression in Murine LiverTissue

To examine the metabolic activity of the FGF19^(K149A) mutant in vivo,6- to 8-week old C57BL/6 mice were fasted overnight and then givenintraperitoneally a single dose (1 mg kg body weight⁻¹) of FGF19^(K149A)or FGF19 as a control. 6 h after the injection, the mice weresacrificed, and liver tissue was excised and frozen. Total RNA wasisolated from liver tissue, and mRNA levels of cholesterol7α-hydroxylase (CYP7A1) and sterol 12α-hydroxylase (CYP8B1) weremeasured using quantitative real time RT-PCR as described previously(Inagaki et al., Cell Metab. 2:217-225 (2005); Kim et al., J. Lipid Res.48:2664-2672 (2007), which are hereby incorporated by reference in theirentirety). The Institutional Animal Care and Use Committee at theUniversity of Texas Southwestern Medical Center at Dallas had approvedthe experiments.

Example 6—Measurement of Serum Phosphate in Mice

The metabolic activity of the FGF23^(R140A/R143A) mutant was examinedboth in normal mice and in Fgf23 knockout mice. 4- to 5-week old C57BL/6mice were given intraperitoneally a single dose (0.29 mg kg bodyweight⁻¹) of FGF23^(R140/R143A) or FGF23 as a control. Before theinjection and 8 h after the injection, blood was drawn from the cheekpouch and spun at 3,000×g for 10 min to obtain serum. Phosphateconcentration in serum was measured using the Phosphorus Liqui-UV Test(Stanbio Laboratory). 6- to 8-week old Fgf23 knockout mice (Sitara etal., Matrix Biol. 23:421-432 (2004), which is hereby incorporated byreference in its entirety) (56) were given two injections ofFGF23^(R140A/R143A) or FGF23 at 8 h intervals (0.71 mg kg body weight⁻¹each), and blood samples were collected for phosphate analysis beforethe first injection and 8 h after the second injection.

To test whether the FGF2^(ΔHBScore)-FGF23^(C-tail) chimera exhibitsFGF23-like metabolic activity, 5- to 6-week old C57BL/6 mice were givena single injection of FGF2^(ΔHBScore)-FGF23^(C-tail) (0.21 mg kg bodyweight⁻¹). As controls, mice were injected withFGF2^(WTcore)-FGF23^(C-tail) or FGF23. Before the injection and 8 hafter the injection, blood samples were collected for measurement ofserum phosphate. To confirm that αKlotho is required for the metabolicactivity of the FGF2^(ΔHBScore)-FGF23^(C-tail) chimera, 7- to 8-week oldαKlotho knockout mice (Lexicon Genetics) were injected once withFGF2^(ΔHBScore)-FGF23^(C-tail) or FGF23 as a control (0.51 mg kg bodyweight⁻¹). Before the injection and 8 h after the injection, bloodsamples were collected for phosphate analysis. The Harvard UniversityAnimal Care and Research committee board had approved all theexperiments.

Example 7—Analysis of CYP27B1 mRNA Expression in Murine Renal Tissue

The ability of the FGF2^(HBScore)-FGF23^(C-tail) chimera to reduce renalexpression of 25-hydroxyvitamin D₃ 1α-hydroxylase (CYP27B1) was used asanother readout for FGF23-like metabolic activity. C57BL/6 mice injectedwith FGF2^(ΔHBScore)-FGF23^(C-tail), FGF2^(WTcore)-FGF23^(C-tail), orFGF23 were sacrificed 8 h after the protein injection, and renal tissuewas excised and frozen. CYP27B1 mRNA levels in total renal tissue RNAwere measured using real time quantitative PCR as described previously(Nakatani et al., FASEB J. 23:3702-3711 (2009); Ohnishi et al., KidneyInt. 75:1166-1172 (2009), which are hereby incorporated by reference intheir entirety). The Harvard University Animal Care and Researchcommittee board had approved the experiments.

Example 8—Insulin Tolerance Test in Mice

The ability of the FGF2^(ΔHBScore)-FGF21^(C-tail) chimera to potentiatethe hypoglycemic effect of insulin was used as readout for FGF21-likemetabolic activity (Ohnishi et al., FASEB J. 25:2031-2039 (2011), whichis hereby incorporated by reference in its entirety). 8- to 12-week oldC57BL/6 mice were kept on normal chow. On the day of the insulintolerance test, mice were fasted for 4 h and then bled from the cheekpouch for measuring fasting blood glucose levels. Thereafter, mice wereadministered intraperitoneally insulin (0.5 units kg body weight⁻¹)alone or insulin (0.5 units-kg body weight⁻¹) plusFGF2^(ΔHBScore)-FGF21^(C-tail) chimera (0.3 mg kg body weight⁻¹). As acontrol, mice were co-injected with insulin plus FGF21. At the indicatedtime points after the injection (FIG. 7G), blood was drawn from the tailvein. Glucose concentrations in the blood samples were determined usingBayer Contour® blood glucose test strips (Bayer Corp.). The HarvardUniversity Animal Care and Research committee board had approved theexperiments.

Example 9—Analysis of Blood Glucose in Ob/Ob Mice

ob/ob mice were injected subcutaneously with FGF1^(ΔNT), FGF1^(ΔHBS), orFGF1^(ΔHBScore)-FGF21^(C-tail) chimera. Injection of native FGF1 ornative FGF21 served as controls. A single bolus of 0.5 mg of protein perkg of body weight was injected. This dose was chosen on the basis thatmaximal efficacy of the hypoglycemic effect of native FGF1 is seen atthis dose. Before the protein injection and at the indicated time pointsafter the injection (FIGS. 9A-9C), blood glucose concentrations weremeasured using an OneTouch Ultra glucometer (Lifescan). TheInstitutional Animal Care and Use Committee at the Salk Institute forBiological Sciences at La Jolla had approved the experiments.

Example 10—Statistical Analysis

Data are expressed as mean±SEM. A Student's t test or analysis ofvariance (ANOVA) was used as appropriate to make statisticalcomparisons. A value of P<0.05 was considered significant.

Example 11—HS is Dispensable for the Metabolic Activity of FGF19 andFGF23

In order to engineer endocrine FGFs devoid of HS binding, the FGF19crystal structure (PDB ID: 2P23; (Goetz et al., Mol. Cell Biol.27:3417-3428 (2007), which is hereby incorporated by reference in itsentirety) was compared with that of FGF2 bound to a heparinhexasaccharide (PDB ID: 1FQ9; (Schlessinger et al., Mol. Cell 6:743-750(2000), which is hereby incorporated by reference in its entirety)).This analysis shows that solvent-exposed residues K149, Q150, Q152, andR157 of FGF19 lie at the corresponding HS-binding site of this ligand,and hence could account for the residual HS binding of FGF19 (FIGS. 1A,1B, and 2). Likewise, comparative analysis of the FGF23 crystalstructure (PDB ID: 2P39; (Goetz et al., Mol. Cell Biol. 27:3417-3428(2007), which is hereby incorporated by reference in its entirety)) withthat of heparin-bound FGF2 (PDB ID: 1FQ9; (Schlessinger et al., Mol.Cell 6:743-750 (2000), which is hereby incorporated by reference in itsentirety)) points to R48, N49, R140, and R143 as candidates mediatingthe residual HS binding of this ligand (FIGS. 1A, 1C, and 2). Inagreement with the structural predictions, replacement of K149 alone inFGF19 with alanine and combined substitution of R140 and R143 in FGF23for alanine were sufficient to abolish residual HS binding of theseligands (FIGS. 3B-3E).

To test the impact of knocking out residual HS binding of FGF19 on thesignaling by this ligand, H4IIE hepatoma cells were stimulated with theFGF19^(K149A) mutant or wild-type FGF19. H4IIE cells endogenouslyexpress FGFR4 and βKlotho (Kurosu et al., J. Biol. Chem. 282:26687-26695(2007), which is hereby incorporated by reference in its entirety), thecognate receptor and co-receptor, respectively, for FGF19. TheFGF19^(K149A) mutant was as effective as wild-type FGF19 in inducingtyrosine phosphorylation of FRS2α and downstream activation of MAPkinase cascade (FIG. 4A). These data show that elimination of residualHS binding has no impact on the ability of FGF19 to signal in culturedcells. To test whether the same holds true for FGF23 signaling, HEK293cells, which naturally express two of the three cognate receptors ofFGF23, namely FGFR1c and FGFR3c (Kurosu et al., J. Biol. Chem.281:6120-6123 (2006), which is hereby incorporated by reference in itsentirety) were transfected with the transmembrane isoform of αKlotho,the co-receptor of FGF23. These cells were treated with theFGF23^(R140A/R143A) double mutant or wild-type FGF23. TheFGF23^(R140A/R143A) mutant had the same capacity as wild-type FGF23 ininducing phosphorylation of FRS2α and downstream activation of MAPkinase cascade (FIG. 4B). These data show that similar to FGF19, FGF23does not need to bind HS in order to activate FGFR in cultured cells.

To substantiate the findings in cells, the metabolic activity ofwild-type and mutated ligands in vivo were compared. Mice were injectedwith the FGF19^(K149A) mutant or wild-type FGF19 and liver geneexpression of CYP7A1 and CYP8B1, which are key enzymes in the major bileacid biosynthetic pathway (Russell, D. W., Annu. Rev. Biochem.72:137-174 (2003), which is hereby incorporated by reference in itsentirety), was analyzed. Like wild-type FGF19, the FGF19^(K149A) mutantmarkedly decreased CYP7A1 and CYP8B1 mRNA levels (FIG. 4C),demonstrating that knockout of residual HS binding does not affect themetabolic activity of FGF19. To examine whether residual HS binding isalso dispensable for the metabolic activity of FGF23, mice were injectedwith the FGF23^(R140A/R143A) mutant or wild-type FGF23 and serumphosphate concentrations were measured. The FGF23^(R140A/R143A) mutantreduced serum phosphate as effectively as wild-type FGF23 (FIG. 4D).Moreover, when injected into Fgf23 knockout mice, theFGF23^(R140A/R143A) mutant exhibited as much of phosphate-loweringactivity as wild-type FGF23 (FIG. 4D). These data show that, as in thecase of FGF19, abolishment of residual HS binding does not impact themetabolic activity of FGF23 leading to the conclusion that HS is not acomponent of the endocrine FGF signal transduction unit (FIG. 1D).

Example 12—Conversion of a Paracrine FGF into an Endocrine LigandConfirms that HS is Dispensable for the Metabolic Activity of EndocrineFGFs

If HS is dispensable for the metabolic activity of endocrine FGFs, thenit should be feasible to convert a paracrine FGF into an endocrine FGFby eliminating HS-binding affinity of the paracrine FGF and substitutingits C-terminal tail for that of an endocrine FGF containing the Klothoco-receptor binding site. Reducing HS-binding affinity will allow theligand to freely diffuse and enter the blood circulation while attachingthe C-terminal tail of an endocrine FGF will home the ligand into itstarget tissues. FGF2, a prototypical paracrine FGF, was chosen forconversion into FGF23-like and FGF21-like ligands, respectively. FGF2was selected as paracrine ligand for this protein engineering exercisebecause it preferentially binds to the “c” isoform of FGFR1, theprincipal receptor mediating the metabolic activity of FGF23 (Gattineniet al., Am. J. Physiol. Renal Physiol. 297:F282-291 (2009); Liu et al.,J. Am. Soc. Nephrol. 19:2342-2350 (2008), which are hereby incorporatedby reference in their entirety) and FGF21 (Kurosu et al., J. Biol. Chem.282:26687-26695 (2007), which is hereby incorporated by reference in itsentirety), respectively. In the crystal structure of heparin-bound FGF2(PDB ID: 1FQ9; (Schlessinger et al., Mol. Cell 6:743-750 (2000), whichis hereby incorporated by reference in its entirety)), K128, R129, andK134 mediate the majority of hydrogen bonds with heparin and hencemutation of these residues was predicted to cause a major reduction inHS-binding affinity of FGF2 (FIGS. 1A, 2, and 5A). Accordingly, thesethree residues were mutated and then the short C-terminal tail of themutated FGF2 was replaced with the C-terminal tail of FGF23 (R161 toI251) or the C-terminal tail of FGF21 (P168 to S209) (FIG. 5A). Theresulting chimeras were termed FGF2^(ΔHBScore)-FGF23^(C-tail) andFGF2^(ΔHBScore)-FGF21^(C-tail) (FIG. 5A). To demonstrate that reductionin HS-binding affinity is required for converting FGF2 into an endocrineligand, two control chimeras were made in which the HS-binding site ofthe FGF2 core was left intact (FGF2^(WTcore)-FGF23^(C-tail) andFGF2^(WTcore)-FGF21^(C-tail); FIG. 5A).

Consistent with the structural prediction,FGF2^(ΔHBScore)-FGF23^(C-tail) and FGF2^(ΔHBScore)-FGF21^(C-tail)exhibited poor binding affinity for HS compared to the correspondingcontrol chimeras with intact HS-binding site (FIGS. 5B-5E). Since HS isan obligatory cofactor in paracrine FGF signaling, theFGF2^(ΔHBScore)-FGF23^(C-tail) and FGF2^(ΔHBScore)-FGF21^(C-tail)chimeras were predicted to lose the ability to activate FGFR1c in anHS-dependent fashion. To test this, HEK293 cells, which endogenouslyexpress FGFR1c, were stimulated with FGF2^(ΔHBScore)-FGF23^(C-tail) orFGF2^(WTcore)-FGF23^(C-tail). Induction of protein expression of thetranscription factor Egr1, a known downstream mediator of FGF signaling,was used as readout for FGFR activation. As shown in FIG. 5G, theFGF2^(ΔHBScore)-FGF23^(C-tail) chimera, like native FGF23, wasineffective in inducing Egr1 expression at concentrations at which theFGF2^(WTcore)-FGF23^(C-tail) chimera elicited a near maximal effect. Thesame observations were made for the FGF2^(ΔHBScore)-FGF21^(C-tail)chimera (FIG. 5F). These data show that, similar to native FGF23 andFGF21, the FGF2^(ΔHBScore)-FGF23^(C-tail) andFGF2^(ΔHBScore)-FGF21^(C-tail) chimeras lost the ability to activateFGFR in an HS-dependent, paracrine fashion.

To determine whether the FGF2^(ΔHBScore)-FGF23^(C-tail) andFGF2^(ΔHBScore)-FGF21^(C-tail) chimeras gained the ability to signal ina Klotho co-receptor-dependent, endocrine fashion, it was first analyzedwhether these chimeras can form ternary complexes with FGFR1c and Klothoco-receptor. To this end, a SPR-based binding competition assay wasemployed. FGF23 was immobilized onto a SPR biosensor chip, and mixturesof a fixed concentration of binary αKlotho-FGFR1c complex withincreasing concentrations of FGF2^(ΔHBScore)-FGF23^(C-tail) chimera werepassed over the chip. FGF2^(ΔHBScore)-FGF23^(C-tail) competed, in adose-dependent fashion, with immobilized FGF23 for binding to theαKlotho-FGFR1c complex (FIG. 7A), demonstrating that the chimera, likenative FGF23 (FIG. 7B), is able to form a ternary complex with FGFR1cand αKlotho. To test whether the FGF2^(ΔHBScore)-FGF21^(C-tail) chimeracan likewise form a ternary complex with FGFR1c and βKlotho, FGF21 wascoupled to a SPR biosensor chip, and mixtures of the binaryβKlotho-FGFR1c complex with FGF2^(ΔHBScore)-FGF21^(C-tail) were passedover the chip. FGF2^(ΔHBScore)-FGF21^(C-tail) effectively competed withimmobilized FGF21 for binding to the βKlotho-FGFR1c complex (FIG. 8A),demonstrating that the chimera, like native FGF21 (FIG. 8B), is capableof binding to the binary complex of FGFR1c and βKlotho. Notably, nativeFGF2 failed to compete with FGF23 for binding to the αKlotho-FGFR1ccomplex (FIG. 7C), and with FGF21 for binding to the βKlotho-FGFR1ccomplex (FIG. 8C) since it lacks the Klotho co-receptor binding domain.To further confirm the binding specificity of theFGF2^(ΔHBScore)-FGF23^(C-tail) chimera for the αKlotho-FGFR1c complex,FGF2^(ΔHBScore)-FGF23^(C-tail) and βKlotho-FGFR1c complex were mixed ata molar ratio of 10:1, and the mixture was injected over a chipcontaining immobilized FGF21. FGF2^(ΔHBScore)-FGF23^(C-tail), likenative FGF23, failed to compete with FGF21 for binding to theβKlotho-FGFR1c complex (FIGS. 7D and 7E). Similarly, theFGF2^(ΔHBScore)-FGF21^(C-tail) chimera, like native FGF21, failed tocompete with FGF23 for binding to the αKlotho-FGFR1c complex (FIGS. 8Dand 8E). For the FGF2^(ΔHBScore)-FGF21^(C-tail) chimera, we investigatedwhether it is able to activate FGFR1c in a βKlotho-dependent fashion incells. HEK293 cells were transfected with βKlotho and then stimulatedwith FGF2^(ΔHBScore)-FGF21^(C-tail) or FGF21. Similar to native FGF21,the FGF2^(ΔHBScore)-FGF21^(C-tail) chimera induced Egr1 proteinexpression in HEK293-βKlotho cells (FIG. 8F), indicating that thechimera is capable of activating FGFR1c in the presence of βKlotho.

To provide definite proof for the ligand conversion, the metabolicactivity of the chimeras in vivo was tested. Specifically, the abilityof the FGF2^(ΔHBScore)-FGF23^(C-tail) chimera to lower serum phosphateand to reduce renal gene expression of CYP27B1, which catalyzes theconversion of vitamin D into its bioactive form, was examined. Mice wereinjected with FGF2^(ΔHBScore)-FGF23^(C-tail) or as controls, FGF23 orFGF2^(WTcore)-FGF23^(C-tail), and serum phosphate concentrations andrenal CYP27B1 mRNA levels were measured. Similar to native FGF23, theFGF2^(ΔHBScore)-FGF23^(C-tail) chimera caused a decrease in serumphosphate in wild-type mice (FIG. 7F). The chimera also induced a markeddecrease in CYP27B1 mRNA levels, just like the native FGF23 ligand (FIG.7G). These data show that the FGF2^(HBScore)-FGF23^(C-tail) chimera actsas an FGF23-like hormone. Importantly, the FGF2^(WTcore)-FGF23^(C-tail)chimera failed to decrease serum phosphate or CYP27B1 mRNA levels (FIGS.7F and 7G). This is expected because, owing to its high affinity for HS,this chimera should be trapped in the vicinity of the injection site andhence not be able to enter the blood circulation. Moreover, these datashow that adding the Klotho co-receptor binding site is not sufficientto convert a paracrine FGF into an endocrine ligand. To confirm that themetabolic activity of the FGF2^(ΔHBScore)-FGF23^(C-tail) chimera isdependent on αKlotho, αKlotho knockout mice were injected withFGF2^(ΔHBScore)-FGF23^(C-tail) or FGF23 as a control, and serumconcentrations of phosphate were measured. As shown in FIG. 7F,FGF2^(ΔHBScore)-FGF23^(C-tail) failed to lower serum phosphate,demonstrating that the chimera, like native FGF23 (FIG. 7F), requiresαKlotho for metabolic activity.

To determine whether the FGF2^(ΔHBScore)-FGF21^(C-tail) chimera exhibitsFGF21-like metabolic activity, its ability to potentiate thehypoglycemic effect of insulin was examined (Ohnishi et al., FASEB J.25:2031-2039 (2011), which is hereby incorporated by reference in itsentirety). Mice were injected with insulin plusFGF2^(ΔHBScore)-FGF21^(C-tail), insulin plus FGF21, or insulin alone,and blood glucose concentrations were monitored for up to one hour afterthe injection. Similar to FGF21, the FGF2^(ΔHBScore)-FGF21^(C-tail)chimera enhanced the hypoglycemic effect of insulin (FIG. 8G),demonstrating that the chimera acts as an FGF21-like hormone.

To substantiate further the concept of FGF ligand conversion, anotherFGF21-like ligand was engineered using FGF1 as paracrine FGF, and themetabolic activity of the engineered protein was tested in vivo in amouse model of diabetes and obesity. Besides serving as an additionalproof-of-concept, the use of FGF1 for this particular ligand conversionwas appealing because FGF1 on its own plays an essential role in glucosemetabolism (Jonker et al., “A PPARγ-FGF1 Axis is Required for AdaptiveAdipose Remodelling and Metabolic Homeostasis,” Nature 485:391-394(2012), which is hereby incorporated by reference in its entirety).Notably, similar to FGF21, FGF1 is induced postprandially in gonadalwhite adipose tissue by the nuclear hormone receptor PPARγ (peroxisomeproliferator activated receptor-γ) (Jonker et al., “A PPARγ-FGF1 Axis isRequired for Adaptive Adipose Remodelling and Metabolic Homeostasis,”Nature 485:391-394 (2012); Dutchak et al., “Fibroblast Growth Factor-21Regulates PPARγ Activity and the Antidiabetic Actions ofThiazolidinediones,” Cell 148:556-567 (2012), which are herebyincorporated by reference in their entirety). FGF1 is required for theremodeling of adipose tissue to adjust to fluctuations in nutrientavailability (Jonker et al., “A PPARγ-FGF1 Axis is Required for AdaptiveAdipose Remodelling and Metabolic Homeostasis,” Nature 485:391-394(2012), which is hereby incorporated by reference in its entirety), andthis process is influenced by FGF21 (Hotta et al., “Fibroblast GrowthFactor 21 Regulates Lipolysis in White Adipose Tissue But is NotRequired for Ketogenesis and Triglyceride Clearance in Liver,”Endocrinology 150:4625-4633 (2009); Dutchak et al., “Fibroblast GrowthFactor-21 Regulates PPARγ Activity and the Antidiabetic Actions ofThiazolidinediones,” Cell 148:556-567 (2012), which are herebyincorporated by reference in their entirety). As part of a positivefeedback loop, FGF21 stimulates PPARγ activity in adipocytes (Dutchak etal., “Fibroblast Growth Factor-21 Regulates PPARγ Activity and theAntidiabetic Actions of Thiazolidinediones,” Cell 148:556-567 (2012),which is hereby incorporated by reference in its entirety), raising theintriguing possibility that FGF21 regulates FGF1 signaling in adiposetissue through PPARγ. An FGF1^(ΔHBScore)-FGF21^(C-tail) chimera wasgenerated in the same manner as the FGF2^(ΔHBScore)-FGF21^(C-tail)chimera (FIGS. 5 and 6). Specifically, K127, K128, and K133 of FGF1,which correspond to the key HS-binding residues identified in thecrystal structure of heparin-bound FGF2 (PDB ID: 1FQ9; (Schlessinger etal., Mol. Cell 6:743-750 (2000), which is hereby incorporated byreference in its entirety)), were mutated and then the short C-terminaltail of the mutated FGF1 was replaced with the C-terminal tail of FGF21(P168 to S209) (FIG. 6). A full-length FGF1 protein harboring theHS-binding site mutations was used as a control (FIG. 6). Consistentwith the structural prediction, this protein exhibited poor bindingaffinity for HS compared to wild-type FGF1 as evidenced by the factthat, unlike the wild-type ligand, the mutant protein did not bind to aHeparin sepharose column. A subcutaneous bolus injection of theFGF1^(ΔHBScore)-FGF21^(C-tail) chimera elicited a hypoglycemic effect inob/ob mice (FIG. 9C), demonstrating that the chimera has metabolicactivity. The effect was of similar magnitude as that observed fornative FGF1 (FIG. 9C), which itself has a much greater hypoglycemiceffect in ob/ob mice than native FGF21 (FIG. 9A). The HS-binding sitemutant of FGF1, which was included as a control in these experiments,showed a similar hypoglycemic effect as the wild-type ligand (FIG. 9B),indicating that the loss in HS-binding affinity had no impact on themetabolic activity of FGF1. To alter the receptor-binding specificity ofFGF1 such that FGF1 selectively binds to the “c” splice isoform ofFGFR1, the principal receptor mediating the metabolic activity of FGF21,an N-terminally truncated FGF1 protein was made (FIG. 6). The truncatedFGF1 ligand lacked twenty four residues from the N-terminus includingthe nine residues that are critical for the promiscuous binding of FGF1to both splice isoforms of FGFR1-3 (Beenken et al., “Plasticity inInteractions of Fibroblast Growth Factor 1 (FGF1) N Terminus with FGFReceptors Underlies Promiscuity of FGF1,” J Biol Chem 287(5):3067-3078(2012), which is hereby incorporated by reference in its entirety).Based on the crystal structures of FGF1-FGFR complexes, the truncationwas also predicted to reduce the receptor-binding affinity of FGF1, andhence the ligand's mitogenicity. The truncated FGF1 protein induced asimilar hypoglycemic effect in ob/ob mice as native FGF1 did (FIG. 9B),indicating that the metabolic activity of FGF1 is mediated through the“c” splice isoform of FGFR. Together, these findings provide a startingpoint for engineering FGF1 ligands that have no mitogenicity but thesame or enhanced metabolic activity compared to native FGF1.

The demonstrated ability to convert a paracrine FGF into an endocrineligand by means of reducing HS-binding affinity of the paracrine FGF andadding the Klotho co-receptor binding site substantiates that HS doesnot participate in the formation of the endocrine FGF signaltransduction unit. The dispensability of HS for the metabolic activityof endocrine FGFs has an intriguing implication as to how these FGFshave evolved to become hormones. It appears that these ligands have lostthe requirement to bind HS in order to signal, while acquiring theability to bind Klotho co-receptors, which is necessary to direct theseligands to their target organs.

In the target tissue, Klotho co-receptors constitutively associate withcognate receptors of endocrine FGFs to offset the inherently lowreceptor-binding affinity of endocrine FGFs (FIGS. 10B-10D; Kurosu etal., J. Biol. Chem. 282:26687-26695 (2007); Kurosu et al., J. Biol.Chem. 281:6120-6123 (2006); Ogawa et al., Proc. Nat'l. Acad. Sci. U.S.A.104:7432-7437 (2007); Urakawa et al., Nature 444:770-774 (2006), whichare hereby incorporated by reference in their entirety). This lowbinding affinity is due to the fact that key receptor-binding residuesin the β-trefoil core of endocrine FGFs are replaced by residues thatare suboptimal for receptor binding (Goetz et al., Mol. Cell Biol.27:3417-3428 (2007), which is hereby incorporated by reference in itsentirety). To measure the degree to which Klotho co-receptors enhancethe receptor-binding affinity of endocrine FGFs, SPR experiments wereconducted using FGF23 and FGFR1c and αKlotho co-receptor as an example(see FIGS. 10A-10F). The SPR data show that αKlotho enhances theaffinity of FGF23 for FGFR1c by over 20-fold (FIGS. 10D and 10E). Theaffinity of FGF23 for FGFR1c in the presence of αKlotho is comparable tothat of FGF2 for FGFR1c in the absence of its HS cofactor (FIGS. 10A and10E). It should be noted, however, that HS further increases the bindingaffinity of FGF2 for FGFR1c by at least an order of magnitude(Pantoliano et al., Biochemistry 33:10229-10248 (1994); Roghani et al.,J. Biol. Chem. 269:3976-3984 (1994), which are hereby incorporated byreference in their entirety). Hence, the receptor-binding affinity ofFGF23 in the presence of αKlotho co-receptor still is lower than that ofFGF2 in the presence of HS cofactor. These observations imply that thesignaling capacity of the endocrine FGF signal transduction unit shouldbe weaker than that of the paracrine FGF signaling unit. Indeed,cell-based studies show that even in the presence of their Klothoco-receptor, endocrine FGFs are inferior to paracrine FGFs at activatingFGFR-induced intracellular signaling pathways (Kurosu et al., J. Biol.Chem. 282:26687-26695 (2007); Urakawa et al., Nature 444:770-774 (2006),which are hereby incorporated by reference in their entirety).

The finding that endocrine FGFs do not need to rely on HS for signalinghas another important implication in regard to the role of Klothoco-receptors. Since FGFR dimerization is a prerequisite for FGFsignaling in general, it is proposed that Klotho co-receptors not onlyenhance the binding affinity of endocrine ligand for receptor but alsopromote receptor dimerization upon ligand binding. In other words,Klotho co-receptors must fulfill the same dual role that HS plays insignaling by paracrine FGFs (FIG. 1D). The ligand conversion alsoprovides the framework for the rational design of endocrine FGF-likemolecules for the treatment of metabolic disorders. An FGF23-likemolecule, for example, will be useful for the treatment of inherited oracquired hyperphosphatemia, and an FGF21-like molecule, for example, forthe treatment of type 2 diabetes, obesity, and related metabolicdisorders.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow.

1.-19. (canceled)
 20. A method for treating a subject suffering from adisorder, the method comprising: selecting a subject suffering from thedisorder; providing a chimeric fibroblast growth factor (“FGF”) protein,wherein the chimeric FGF protein comprises an N-terminus coupled to aC-terminus, wherein the N-terminus comprises a portion of an FGF2 andthe C-terminus comprises a C-terminal portion of FGF19, and wherein theportion of the FGF2 is modified to decrease binding affinity for heparinand/or heparan sulfate compared to the portion without the modification;and administering a therapeutically effective amount of the chimeric FGFprotein to the selected subject under conditions effective to treat thedisorder. 21.-22. (canceled)
 23. The method according to claim 20,wherein the portion of the FGF2 comprises an amino acid sequencebeginning at any one of residues 1 to 25 and ending at any one ofresidues 151 to 155 of SEQ ID NO:
 121. 24. The method according to claim23, wherein the portion of the FGF2 comprises amino acid residues 1-151or 25-151 of SEQ ID NO:
 121. 25. The method according to claim 23,wherein the portion of the FGF2 comprises amino acid residues 1-152,1-153, 1-154, 1-155, 2-151, 2-152, 2-153, 2-154, 2-155, 3-151, 3-152,3-153, 3-154, 3-155, 4-151, 4-152, 4-153, 4-154, 4-155, 5-151, 5-152,5-153, 5-154, 5-155, 6-151, 6-152, 6-153, 6-154, 6-155, 7-151, 7-152,7-153, 7-154, 7-155, 8-151, 8-152, 8-153, 8-154, 8-155, 9-151, 9-152,9-153, 9-154, 9-155, 10-151, 10-152, 10-153, 10-154, 10-155, 11-151,11-152, 11-153, 11-154, 11-155, 12-151, 12-152, 12-153, 12-154, 12-155,13-151, 13-152, 13-153, 13-154, 13-155, 14-151, 14-152, 14-153, 14-154,14-155, 15-151, 15-152, 15-153, 15-154, 15-155, 16-151, 16-152, 16-153,16-154, 16-155, 17-151, 17-152, 17-153, 17-154, 17-155, 18-151, 18-152,18-153, 18-154, 18-155, 19-151, 19-152, 19-153, 19-154, 19-155, 20-151,20-152, 20-153, 20-154, 21-155, 21-151, 21-152, 21-153, 21-154, 21-155,22-151, 22-152, 22-153, 22-154, 22-155, 23-151, 23-152, 23-153, 23-154,23-155, 24-151, 24-152, 24-153, 24-154, 24-155, 25-152, 25-153, 25-154,or 25-155 of SEQ ID NO:
 121. 26.-27. (canceled)
 28. The method accordingto claim 23, wherein the modification comprises one or moresubstitutions located at one or more amino acid residues of SEQ ID NO:121 selected from the group consisting of N36, K128, R129, K134, K138,Q143, K144, and combinations thereof.
 29. The method according to claim28, wherein the one or more substitutions are selected from the groupconsisting of N36; K128D; R129Q; K134V; K138H; Q143M; K144T, K144L, orK144I; and combinations thereof.
 30. The method according to claim 20,wherein the C-terminal portion comprises a β-Klotho co-receptor bindingdomain.
 31. The method according to claim 20, wherein the C-terminalportion from FGF19 begins at a residue corresponding to any one of aminoacid residues 169, 197, or 204 of SEQ ID NO:
 233. 32. The methodaccording to claim 20, wherein the C-terminal portion from FGF19comprises an amino acid sequence selected from the group consisting ofamino acid residues 204 to 216, amino acid residues 197 to 216, andamino acid residues 169 to 216 of SEQ ID NO:
 233. 33. The methodaccording to claim 20, wherein the C-terminal portion from FGF19comprises an amino acid sequence selected from the group consisting of(SEQ ID NO: 281) TGLEAV(R/N)SPSFEK;  (SEQ ID NO: 282)MDPFGLVTGLEAV(R/N)SPSFEK; and (SEQ ID NO: 283)LP(M/I)(V/A)PEEPEDLR(G/R)HLESD(M/V)FSSPLETDSMDPFGL VTGLEAV(R/N)SPSFEK.


34. The method according to claim 32, wherein the C-terminal portionfrom FGF19 further comprises one or more substitutions, additions, ordeletions while retaining the ability to bind β-Klotho.
 35. The methodaccording to claim 32, wherein the C-terminal portion from FGF19 furthercomprises one or more substitutions, additions, or deletions to enhancebinding affinity for β-Klotho.
 36. The method according to claim 20,wherein the disorder is associated with diabetes, obesity, or metabolicsyndrome.
 37. The method according to claim 36, wherein the disorder istype II diabetes, gestational diabetes, or drug-induced diabetes. 38.The method according to claim 36, wherein the disorder is type Idiabetes.
 39. The method according to claim 36, wherein the disorder isobesity.
 40. The method according to claim 37, wherein the disorder ismetabolic syndrome.
 41. The method according to claim 20, wherein theadministering is performed subcutaneously, intravenously,intramuscularly, intraperitoneally, by intranasal instillation, byimplantation, by intracavitary or intravesical instillation,intraocularly, intraarterially, intralesionally, transdermally, or byapplication to mucous membranes.
 42. The method according to claim 20,wherein the chimeric protein is administered with apharmaceutically-acceptable carrier.
 43. The method according to claim20, wherein the selected subject is a mammal.
 44. The method accordingto claim 20, wherein the selected subject is a human.
 45. The methodaccording to claim 20, wherein the chimeric FGF is co-administered withone or more agents selected from the group consisting of ananti-inflammatory agent, an antifibrotic agent, an antihypertensiveagent, an antidiabetic agent, a triglyceride-lowering agent, and acholesterol-lowering agent.
 46. A method of making a chimeric fibroblastgrowth factor (“FGF”) protein possessing enhanced endocrine activity,the method comprising: introducing one or more modifications to a FGFprotein, wherein the modification decreases the affinity of the FGFprotein for heparin and/or heparan sulfate; and coupling a C-terminalportion of FGF19 comprising a 13-Klotho co-receptor binding domain tothe modified FGF protein's C-terminus, whereby a chimeric FGF proteinpossessing enhanced endocrine activity is made. 47.-60. (canceled)
 61. Amethod of facilitating fibroblast growth factor receptor(“FGFR”)-βKlotho complex formation, the method comprising: providing acell comprising βKlotho co-receptor and an FGFR; providing a chimericfibroblast growth factor (“FGF”) protein comprising a C-terminal portionof FGF19 and a portion of a paracrine FGF, wherein the portion of theparacrine FGF is modified to decrease binding affinity for heparinand/or heparan sulfate compared to the portion without the modification;and contacting the cell with the chimeric FGF protein under conditionseffective to cause FGFR-Klotho co-receptor complex formation. 62.-77.(canceled)
 78. A method of screening for agents capable of facilitatingfibroblast growth factor receptor (“FGFR”)-βKlotho co-receptor complexformation in the treatment of a disorder, the method comprising:providing a chimeric fibroblast growth factor (“FGF”) comprising anN-terminus coupled to a C-terminus, wherein the N-terminus comprises aportion of a paracrine FGF and the C-terminus comprises a C-terminalportion of FGF19, and wherein the portion of the paracrine FGF ismodified to decrease binding affinity for heparin and/or heparan sulfatecompared to the portion without the modification; providing binaryβKlotho-FGFR complex; providing one or more candidate agents; combiningthe chimeric FGF, the binary βKlotho-FGFR complex, and the one or morecandidate agents under conditions permitting the formation of a ternarycomplex between the chimeric FGF and the binary βKlotho-FGFR complex inthe absence of the one or more candidate agents; and identifying the oneor more candidate agents that decrease ternary complex formation betweenthe chimeric FGF and the binary βKlotho-FGFR complex compared to theternary complex formation in the absence of the one or more candidateagents as suitable for treating the disorder. 79.-86. (canceled)